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Modified atmosphere packaging of pink prawn (Pandalus platyceros) Dheeragool, Panadda 1989

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MODIFIED ATMOSPHERE PACKAGING OF PINK PRAWN  (Pandalus  platyceros) By  PANADDA DHEERAGOOL B . S c , Kasetsart U n i v e r s i t y , 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE FACULTY OF GRADUATE STUDIES Department of Food Science We accept t h i s t h e s i s as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA May 1989 © Panadda Dheeragool, 1989  In presenting this thesis in partial fulfilment of the degree at the  study. I further agree that permission for extensive  copying of this thesis for scholarly purposes may or  by  his or  her  representatives.  be  granted by the head of  It is understood  publication of this thesis for financial gain shall not be  Panadda Dheeragool  Department of  Food Science  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6(3/81)  May  1989  that  copying  allowed without my  permission.  Date  advanced  University of British Columbia, I agree that the Library shall make it  freely available for reference and  department  requirements for an  my or  written  ii  ABSTRACT  Pandalus platyceros aerobic (3)  control  nitrogen  (bags  ( p i n k prawn o r s p o t left  atmospheres  open t o  (NMAP)  at  shrimp)  was s t o r e d u n d e r  air),  (2)  carbon dioxide  1°C.  Facultative  (CMAP),  anaerobes  of psychrotrophic  b a c t e r i a w h i c h c o n t a m i n a t e d the prawns  study.  Tissue  pH o f  CMAP p r a w n s  brought  about  the  microorganisms  were  inhibited  under  proteins,  water-soluble  detected  in  the  the  prawns  inosine,  No  and  prawns  was  slower  under  (Hunter  most the  CMAP  the  the  TMA,  time  of  muscle  of  ADP  value).  by  storage  the  decompositions.  than  under  by  the  resulting  Shelf-life  system  NMAP  was  decomposition  NMAP.  Redness  but  no  of  the  pink  shelf-life  colour prawns  extension  in  No ATP was  CMAP  of  IMP  in lighter  slowly  found  Development  caused  slightly  was  AMP d e g r a d e d m o s t  of  the  was  at  was  IMP,  K-value and  in was  prawn  colour  But  carbon  destruction of  prawns  on  rate  likely  the  The  degradation  CMAP  and  and  salt-soluble  NMAP a n d t h e  atmospheres  this  tissue.  t h e NMAP.  packaging.  followed  in  CMAP a n d w e r e  formation  the  prawns  a n d TVB p r o g r e s s e d m o s t  of  influenced  all  t h e NMAP a n d t h e CMAP.  during storage  CMAP  under  of  was m a i n t a i n e d u n d e r  pigments  the  the  prawns  strongly  in  under  at  hypoxanthine  a value)  L  the  Consequently,  proteins,  effect  dioxide  (Hunter  NMAP.  of  lowest  t h e CMAP b u t d e l a y e d u n d e r t h e NMAP.  control  respectively.  inhibited  the  TMA p r o d u c t i o n was f a v o r e d u n d e r  a c c e l e r a t e d under in  exudat i o n  markedly  the  t h e CMAP p r a w n s .  rapidly  greatest  was  and  were  majority  the  (1)  of  stored least  the  prawns doubled  afforded  by  the  NMAP s y s t e m . The  first  accounted for  principal  59.07% o f  the  component  of  data variance.  the This  prawn means  quality  parameter  t h e most  important  iii  q u a l i t y d e s c r i p t o r s of the prawns were a l l m i c r o b i o l o g i c a l v a r i a b l e s , raw prawn meat odour and colour scores, TMA, cooked prawn meat odour and colour scores, water-soluble p r o t e i n and o v e r a l l sensory score. discriminant psychrotrophic concentration  analysis  of  bacterial as  pH count  the grouping  and  data  visually  of  variable,  c l a s s i f i c a t i o n of the prawn samples. v a r i a b l e s were obtained.  total  aerobic the  Two equations  with  TMAN  i n 97.2%  correct  f o r the canonical  A p l o t of these two canonical v a r i a b l e s w i l l  i n d i c a t e the region of prawn q u a l i t y  unacceptable).  sulphide-producing  prawns  resulted  Stepwise  (good, acceptable, or  iv  TABLE OF CONTENTS Page  ABSTRACT LIST OF ABBREVIATIONS LIST OF TABLES LIST OF FIGURES ACKNOWLEDGMENTS 1.  INTRODUCTION  2. LITERATURE REVIEW 2.1. Changes During Storage o f Prawns Under Normal A i r Environment 2.1.1. Change i n Muscle pH 2.1.2. Change i n Exudation Formation 2.1.3. Changes i n ATP and I t s Related Compounds 2.1.4. Change i n T o t a l V i a b l e Psychrotrophic B a c t e r i a l Count 2.1.5. Change i n T o t a l V o l a t i l e Basic Nitrogen Concentration 2.1.6. Change i n Trimethylamine-nitrogen Concentration 2.1.7. Changes i n Concentrations of Water-soluble and S a l t - s o l u b l e Muscle Proteins 2.1.8. Change i n Colour 2.2. Changes Under Carbon Dioxide Modified Atmosphere Storage 2.3. Changes Under Nitrogen Modified Atmosphere Storage 2.4. M u l t i v a r i a t e Analyses 2.4.1. Factor Analysis 2.4.2. Stepwise Discriminant Analysis 3. MATERIALS AND METHODS 3.1. Prawn Samples 3.2. Packaging M a t e r i a l 3.3. Treatments 3.4. Sampling Plans 3.5. Tests 3.5.1. Headspace Gas Compositions i n the Bags 3.5.2. Exudate Determination 3.5.3. M i c r o b i o l o g i c a l Tests 3.5.4. Determination of pHs of Prawn Inner Tissue 3.5.5. K-value Determination 3.5.5.1. Sample Preparation 3.5.5.2. HPLC Conditions 3.5.5.3. R e p r o d u c i b i l i t y of the HPLC Analysis 3.5.5.4. I n t e r n a l Standard 3.5.5.5. R e p r o d u c i b i l i t y of the E x t r a c t i o n 3.5.6. Trimethylamine-nitrogen Determination 3.5.7. Total V o l a t i l e Basic Nitrogen Determination 3.5.8. Sensory Evaluation 3.5.9. Hunter L, a, b Values  i i vi v i i viii x i i 1 6 7 7 8 8 10 11 12 14 15 18 22 22 22 23 25 26 26 28 28 29 30 30 35 35 35 36 37 37 41 41 44 44 57  V  3.5.10. Water-soluble and S a l t - s o l u b l e P r o t e i n Determinations 3.6. S t a t i s t i c a l Analyses 4. RESULTS 4.1. T r i a l 1 4.1.1. Headspace Gas Composition 4.1.2. Exudate Formation 4.1.3. Tissue pH 4.1.4. Microbiology 4.1.5. K-values 4.1.6. Trimethylamine-nitrogen Concentration 4.1.7. Sensory Evaluation 4.1.8. Hunter L, a, b Values 4.2. T r i a l 2 4.2.1. Headspace Gas Composition 4.2.2. Tissue pH 4.2.3. Exudate Formation 4.2.4. Microbiology 4.2.5. Trimethylamine-nitrogen Concentration 4.2.6. T o t a l V o l a t i l e Basic Nitrogen Concentration 4.2.7. Soluble Proteins 4.2.8. Sensory Evaluation 4.2.9. Hunter L, a, b Values 4.2.10. Factor A n a l y s i s 4.2.11. Stepwise Discriminant A n a l y s i s 5.  DISCUSSION 5.1. Headspace Gas Compositions i n the Bags 5.2. Tissue pHs 5.3. Exudate Formation 5.4. T o t a l Psychrotrophic B a c t e r i a l Counts and T o t a l Sulphide-producing Psychrotrophic B a c t e r i a l Counts 5.5. Trimethylamine-nitrogen Concentration 5.6. T o t a l V o l a t i l e Basic Nitrogen Concentration 5.7. K-value and I t s Related Compounds 5.8. S a l t - s o l u b l e P r o t e i n and Water-soluble P r o t e i n Concentrations 5.9. Colour 5.10. Sensory C h a r a c t e r i s t i c s 5.11. S h e l f - l i f e of the Prawns  6. CONCLUSION 7. REFERENCES 8. APPENDIX A: 9. APPENDIX B:  CHLORAMPHENICOL TREATMENT SAMPLE MEANS AND STANDARD DEVIATIONS  57 57 63 64 64 70 70 73 80 83 92 96 99 99 103 108 110 110 114 123 127 131 134 135 135 136 141 142 143 144 145 146 147 148 150 154 163 170  vi  LIST OF ABBREVIATIONS a ADP AMP ANOVA ATP b CC CF CMAP CO CT HPLC Hx HxR IMP I.S. L MAP NMAP OVERALL RC RO TMA TMAN TMAO TSA TSN TVBN SPA SPN SSP WSP  Hunter a value Adenosine diphosphate Adenosine monophosphate Analysis o f variance Adenosine triphosphate Hunter b value Sensory colour score o f cooked prawn meat Sensory flavour score of cooked prawn meat Carbon dioxide modified atmosphere packag(ing/ed) Sensory odour score o f cooked prawn meat Sensory texture score o f cooked prawn meat High performance l i q u i d chromatograph(y/ic) Hypoxanthine Inosine Inosine monophosphate I n t e r n a l standard (5-bromouracil) Hunter L value Modified atmosphere packaging Nitrogen modified atmosphere packag(ing/ed) Mean o f a l l sensory scores: O v e r a l l = (RC+R0+CC+C0+CF+CT)/6 Sensory colour score o f raw prawn meat Sensory odour score o f raw prawn meat Trimethylamine Trimethylamine-nitrogen Trimethylamine oxide Total aerobic psychrotrophic b a c t e r i a l count T o t a l anaerobic psychrotrophic b a c t e r i a l count Total v o l a t i l e b a s i c nitrogen Total aerobic sulphide-producing psychrotrophic b a c t e r i a l count Total anaerobic sulphide-producing psychrotrophic b a c t e r i a l count S a l t - s o l u b l e proteins Water-soluble proteins  vii  LIST OF TABLES Table  Page  1  Properties of DUPONT LP 920 packaging f i l m  27  2  R e p r o d u c i b i l i t y o f the HPLC column  39  3  K-values o f the nucleotide standard solutions w i t h and without the area count o f the small unknown peak o f the i n t e r n a l standard (n=3) 42  4  R e p r o d u c i b i l i t y o f the e x t r a c t i o n f o r K-value determination  43  5  C h a r a c t e r i s t i c changes o f pink prawns during spoilage  46  6  F - s t a t i s t i c s from ANOVA on data of T r i a l 1  68  7  F - s t a t i s t i c s from ANOVA on data of i n d i v i d u a l sampling day of Trial 1  69  8  F - s t a t i s t i c s from Friedman two-way ANOVA on sensory data o f Trial 1  9 10 11  91  F - s t a t i s t i c s from ANOVA on data o f 12 days storage o f T r i a l 2 ... 101 F - s t a t i s t i c s from Friedman two-way ANOVA on sensory data of Trial 2 Sorted rotated f a c t o r loadings from f a c t o r analysis o f data o f Trial 2  122 128  12  Rotated f a c t o r loadings (pattern) from f a c t o r analysis o f data o f Trial 2 129  13  C o r r e l a t i o n matrix from f a c t o r analysis of data o f T r i a l 1  137  14  C o r r e l a t i o n matrix from f a c t o r analysis of data o f T r i a l 2  139  viii  LIST OF FIGURES  Figure  Page  1  Dimensions of the Hunter L, a, b colour coordinate system  17  2  Chromatogram of the headspace gases i n the CMAP bags  31  3  Chromatogram of the headspace gases i n the NMAP bags  32  4  Anaerobic chamber  34  5  Chromatogram of the nucleotide standard working s o l u t i o n  38  6  Chromatogram of the i n t e r n a l standard 5-bromouracil stock solution  40  Sensory evaluation score sheet f o r cooked prawn meat odour, f l a v o u r , and texture scores  48  8  Sensory evaluation score sheet f o r cooked prawn meat colour score  50  9  Sensory evaluation score sheet f o r raw prawn meat odour and colour scores  52  Temperature gradient at the center o f the preheated (200°C) conventional oven  55  7  10 11  Temperature gradient i n s i d e the edible prawn t i s s u e during cooking at 200°C  56  12  M u l t i v a r i a t e data input format  59  13  T r i a l 1: Concentrations of carbon d i o x i d e , oxygen, and nitrogen gas i n the CMAP bags stored at 1°C T r i a l 1: Concentrations o f carbon d i o x i d e , oxygen, and nitrogen gas i n the NMAP bags stored at 1°C  14  65 66  15  T r i a l 1: Exudate volumes o f prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C 67  16  T r i a l 1: Inner t i s s u e pHs o f prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres a t T C  71  T r i a l 1: Total aerobic and t o t a l anaerobic psychrotrophic b a c t e r i a l counts o f prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  72  17  18  T r i a l 1: K-values o f prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C 74  ix  19  20  21  22 23 24  25 26 27  28  29  30 31  32 33  T r i a l 1: Adenosine diphosphate concentrations of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  75  T r i a l 1: Adenosine monophosphate concentrations of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  77  T r i a l 1: Inosine monophosphate concentrations of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  78  T r i a l 1: Inosine concentrations of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  79  T r i a l 1: Hypoxanthine concentrations of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres a t 1°C  81  T r i a l 1: Trimethylamine-nitrogen concentrations of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres a t 1°C '  82  T r i a l 1: O v e r a l l sensory scores of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres a t T C  84  T r i a l 1: Scores f o r raw prawn meat odour of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres a t 1°C  85  T r i a l 1: Scores f o r cooked prawn meat odour of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  86  T r i a l 1: Scores f o r cooked prawn meat colour of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  87  T r i a l 1: Scores f o r cooked prawn meat texture of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  88  T r i a l 1: Scores f o r raw prawn meat colour of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  89  T r i a l 1: Scores f o r cooked prawn meat flavour of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  90  T r i a l 1: Hunter L values of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  93  T r i a l 1: Hunter a values of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  94  X  34  T r i a l 1: Hunter b values of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C 95  35  T r i a l 2: Concentrations o f carbon d i o x i d e , oxygen, and nitrogen gas i n the CMAP bags stored a t 1°C  97  T r i a l 2: Concentrations o f carbon d i o x i d e , oxygen, and nitrogen gas i n the NMAP bags stored at 1°C  98  36 37  T r i a l 2: Inner t i s s u e pHs of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  100  38  T r i a l 2: Exudate volumes o f prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C 102  39  T r i a l 2: T o t a l aerobic psychrotrophic b a c t e r i a l counts o f prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and n i t r o g e n atmospheres a t 1°C 104  40  T r i a l 2: Total anaerobic psychrotrophic b a c t e r i a l counts o f prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C 105  41  T r i a l 2: T o t a l aerobic sulphide-producing psychrotrophic b a c t e r i a l counts o f prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  106  T r i a l 2: T o t a l anaerobic sulphide-producing psychrotrophic b a c t e r i a l counts o f prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  107  42  43  T r i a l 2: Trimethylamine-nitrogen concentrations of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C 109  44  T r i a l 2: Total v o l a t i l e basic nitrogen concentrations o f prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and n i t r o g e n atmospheres a t 1°C I l l  45  T r i a l 2: Water-soluble p r o t e i n concentrations o f prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  112  T r i a l 2: S a l t - s o l u b l e p r o t e i n concentrations of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  113  T r i a l 2: O v e r a l l sensory scores of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  115  46  47 48  T r i a l 2: Scores f o r raw prawn meat colour of prawns stored under aerobic c o n t r o l , carbon dioxide, and nitrogen atmospheres a t 1°C 116  xi  49  T r i a l 2: Scores f o r raw prawn meat odour of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres a t 1°C 117  50  T r i a l 2: Scores f o r cooked prawn meat colour of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  118  T r i a l 2: Scores f o r cooked prawn meat odour of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  119  T r i a l 2: Scores f o r cooked prawn meat flavour of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  120  T r i a l 2: Scores f o r cooked prawn meat texture of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  121  51  52  53  54  T r i a l 2: Hunter L values of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres a t 1°C 124  55  T r i a l 2: Hunter a values of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C 125  56  T r i a l 2: Hunter b values of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C 126  57  T r i a l 2: P l o t s of Factor 1 against Factor 2 of rotated f a c t o r loadings from the f a c t o r a n a l y s i s on data of 12 days storage .... 130  58  T r a i l 2: Canonical p l o t s of prawn samples c l a s s i f i e d prawns by stepwise discriminant a n a l y s i s of pH and SPA  133  xii  ACKNOWLEDGEMENT  I wish to express my sincere gratitude to my thesis supervisor Dr. B.J.  Skura f o r a l l h i s time, patience, guidance, and understanding.  also wish  to thank  the members of my graduate  committee:  I  Drs. W.D.  Powrie, P. Townsley, S. Nakai, and G. Strasdine, f o r a l l t h e i r valuable suggestions and guidance. For t e c h n i c a l assistance and advice, I am g r e a t f u l to Dr. R. Wu, Sherman Yee, and V a l e r i e Skura.  For advice i n s t a t i s t i c a l analyses, I  thank Dr. N. Heckman, L i s a Kan, C a l v i n L a i , and Stanley K i t a . Dr. Eunice Li-Chan f o r assistance  i n using the BMDP program.  I thank I am  g r e a t f u l to Seema Datta and Sunny Lee f o r t h e i r help i n packaging 150 l b s of prawns and f o r proofreading t h i s t h e s i s . their  enthusiastic  participation.  My  I thank my sensory panel f o r  appreciation  also  goes  to the  departmental s e c r e t a r i e s Joyce Tom and Charlene Campbell. Very  special  thanks  goes  to Murray  Willing  f o r h i s endless  encouragement and supports, and f o r making my stay i n Canada the most wonderful thing i n my l i f e .  My gratitude also goes to h i s l o v i n g parents  and t h e i r family members who always make me f e e l so much at home. I thank both the Canadian and Thai Governments f o r arranging t h i s scholarship  program  Technology P r o j e c t .  under  the ASEAN-CANADA  Fisheries  Post-Harvest  INTRODUCTION  2  1.  INTRODUCTION  Q u a l i t y d e t e r i o r a t i o n of a fresh number of d i f f e r e n t causing  factors.  off-odours which  are  f i s h e r y product r e s u l t s  Bacterial associated  action with  from a  releases metabolites spoilage.  Therefore,  c o n t r o l of b a c t e r i a l a c t i v i t y i s the key to extend the s h e l f - l i f e of the product. Applications  of  modified atmosphere  packaging  (MAP)  systems  f i s h e r y products were f i r s t published by Coyne (1932 and 1933).  on  Carbon  dioxide storage atmosphere was found to extend s h e l f - l i f e of the f i s h at 15°C and s h e l f - l i f e was further extended when the storage temperature was at 2°C. A modified atmosphere packaging (MAP) in  which  differs  the composition of the gaseous from the composition of normal  system i s an enclosed system environment  air.  The  normally predetermined at the time of packaging.  i n the package  gas  composition i s  Once the package i s  sealed, there i s no external c o n t r o l of the atmosphere. films  used  f o r modified atmosphere  packages  (MAP)  The  usually  plastic have  gas  b a r r i e r properties to help maintain atmospheric composition w i t h i n the packages i n a desired range. Most of the storage atmospheres applied to f i s h e r y products were p r i m a r i l y enriched with carbon dioxide gas, as a means of c o n t r o l l i n g b a c t e r i a l growth, r e s u l t i n g i n the preservation of the  products.  Commercially, MAP  system f o r f i s h e r y products employs a  gas mixture of carbon dioxide and nitrogen rather than carbon dioxide alone since carbon dioxide dissolves i n the aqueous phase of the product tissue and causes package collapse (Lannelongue et al.,  1982).  Further,  3  carbon dioxide causes a s i g n i f i c a n t weight loss of the product due to the formation of large amounts of exudate. Besides the lengthening the s h e l f - l i f e o f the f i s h e r y product, the MAP system has other advantages  such as: improved q u a l i t y  maintenance,  extended t r a n s i t time, l e s s problems with melting i c e , b e t t e r access to the r e t a i l market, a c t i v e i n h i b i t i o n of b a c t e r i a l and fungal growth, and reduced  economic  include:  added  losses cost;  personnel t r a i n i n g  (Wolfe,  variable  1980; Smith, product  1987).  Disadvantages  requirements;  equipment and  requirements; need f o r good q u a l i t y  raw m a t e r i a l ,  maintenance o f package i n t e g r i t y , and good temperature c o n t r o l (Wolfe, 1980; Smith, 1987). constantly Clostridium  Storage temperatures below 2°C (Smith, 1987) must be  maintained botulinum  to  prevent  the  growth  whose t o x i n can cause botulism.  of  non-proteolytic  This t o x i n i s heat  s e n s i t i v e and i s destroyed when the product i s cooked p r o p e r l y . Pink prawn,  Pandalus  platyceros,  harvested i n B r i t i s h Columbia.  i s the l a r g e s t o f the l o c a l prawns  I t now ranks f i r s t i n landed value i n the  prawn f i s h e r y (Department of F i s h e r i e s and Oceans, Canada/1359 UW/29E). The common name f o r  Pandalus  U.S.A., i s spot shrimp. d a l i d shrimp.  recognized by the F.A.O. and the  platyceros,  I t i s also larger than most o f the P a c i f i c pan-  The species inhabits the Northern P a c i f i c Ocean from the  Bering S t r a i t s to Japan and Korea i n the west, to Southern C a l i f o r n i a i n the east (Dore and Frimodt, 1987).  I t s body c o l o r i s u s u a l l y reddish  brown or tan with white h o r i z o n t a l bars on the carapace, and has the distinctive  white  spots  on the f i r s t  and f i f t h  abdominal  segments  (Department o f F i s h e r i e s and Oceans/1359 UW/29E). Previous studies on headless, s h e l l - o n penaeid shrimps r e f r i g e r a t i o n temperature, under a i r environment  stored at  (Cobb et a l . , 1973; and  4  Lannelongue et a.1., 1982) showed that the s h e l f - l i f e of shrimp from the Gulf of Mexico was only about a week or l e s s . enriched  atmosphere  temperature  of  as  the  4°C, the  (Lannelongue et al., 1982).  storage  By using a carbon dioxide  atmosphere  shelf-life  of  the  at  shrimp  refrigeration was  extended  One hundred percent carbon dioxide as the  storage atmosphere doubled the s h e l f - l i f e of these shrimps to 14 days. This extended s h e l f - l i f e i s b e n e f i c i a l .  Since the prawns are a v a i l a b l e  only 6 months of the year, the demand f o r t h i s prawn species i s great, and  the p r i c e  i s h i g h , extended s h e l f - l i f e  means a longer  marketing  p e r i o d . The extended s h e l f - l i f e also makes t r a n s p o r t a t i o n of the prawns to remote areas i n f r e s h form p o s s i b l e . Up to the present time, more information on the e f f e c t s of carbon dioxide and nitrogen as storage atmospheres on the q u a l i t y of the prawns is  still  freshness  needed.  In the marketplace,  i t i s essential  to have some  or spoilage i n d i c a t o r ( s ) that can be r e l i a b l y used to help  e s t a b l i s h the q u a l i t y of the prawns as w e l l as other f i s h e r y that were stored under a MAP system.  products  I t i s also very important  to be  able to use these spoilage i n d i c a t o r s as a means to p r e d i c t spoilage as w e l l as s h e l f - l i f e l i m i t of the prawns.  Several i n v e s t i g a t i o n s on MAP  storage of f i s h e r y products used t o t a l b a c t e r i a l count, TVBN, and TMAN concentrations shrimp  to evaluate changes i n many f i s h e r y products  including  (Lannelongue et al., 1982; Parkin et al., 1982; L a y r i s s e amd  Matches, 1984).  However, other i n d i c a t o r s , such as K-value, Hunter L, a,  b values, and sulphide-producing been explored.  psychrotrophic b a c t e r i a have not yet  5  The objectives of this research were: (1) to measure the effects of carbon dioxide modified atmosphere packaging  (CMAP) and nitrogen  modified atmosphere  packaging  (NMAP)  systems on the microbial, chemical, and sensory changes in the pink prawn; and (2) to statistically determine an indicator or a combination of indicators that can be effectively used for predicting the spoilage or shelf-life of pink prawns.  LITERATURE REVIEW  7  2.  2.1  LITERATURE REVIEW  Changes during storage of prawns under normal a i r environment. The process of f i s h spoilage has been found to be rather complicated  as i t i s caused by a number of i n t e r r e l a t e d systems.  In general, a f t e r a  f i s h d i e s , i t s r e s p i r a t i o n and thus i t s oxygen supply stops.  Spoilage  passes through the stages of r i g o r m o r t i s , d i s s o l u t i o n of r i g o r m o r t i s , and a u t o l y s i s r e s p e c t i v e l y . glycogen by  tissue  G l y c o l y s i s proceeds with the breakdown of  enzymes followed by  through a s e r i e s of r e a c t i o n s . pH  drop  i n the muscle and  the formation of l a c t i c  The accumulation of l a c t i c a c i d causes a  induces  the  liberation  inherent a c i d proteases.  Proteolytic activities  microbial  i n the  enzymes r e s u l t  ATP  struggle  after  hypoxanthine  immmediately accumulation.  and  activation  of  of t i s s u e enzymes and  formation of non-protein nitrogenous  compounds and amino a c i d s . and  acid  degradation proceeds death,  eventually  during the death resulting  in  These a u t o l y t i c products support the growth  of microorganisms that contaminate the animal.  A number of v o l a t i l e , low  molecular weight compounds are formed as a r e s u l t of m i c r o b i a l actions and contributed to spoilage off-odour. During storage under normal a i r environment, changes that occur after the death of the f i s h that have been used to i n d i c a t e q u a l i t y of the edible tissues are reviewed below. 2.1.1.  Change i n muscle pH  G l y c o l y s i s i s the process i n which glycogen i s broken-down to l a c t i c a c i d and as a consequence, t i s s u e pH i s lowered.  Even though the pattern  of decrease of glycogen and increase of l a c t i c a c i d i n shrimp are s i m i l a r  8  to those of other marine and land animals ( F l i c k and L o v e l l , 1972), i n many studies on shrimp, the absence of an i n i t i a l postmortem drop i n pH has also been reported (Layrisse and Matches, 1984; F l i c k and L o v e l l , 1972; B a i l e y et al. , 1956). muscle.  This may be one unique feature o f shrimp  I t may be because the glycogen l e v e l s i n shrimp are lower than  those i n f i s h ; or i t may be associated with a release of low molecular weight  bases  i n the t i s s u e  soon  after  death, as crustacean  muscle  contains higher concentrations of non-protein nitrogenous compounds than f i s h and mammals ( F l i c k and L o v e l l , 1972). 2.1.2.  Change i n exudation formation  This  decreasing pH causes  some of the sarcoplasmic proteins to  aggregate and form a p r e c i p i t a t e . onto  myofibrillar  proteins  causes  The p r e c i p i t a t i o n of these proteins the s o l u b i l i t y  and water-holding  capacity of m y o f i b r i l l a r proteins to decrease (Khan, 1977). As a r e s u l t of the decreasing water-holding capacity o f the myofib r i l l a r p r o t e i n s , a p o r t i o n o f water separates from the muscle  cells.  This p o r t i o n o f water i s c a l l e d exudate or d r i p . In porcine muscle, a pH lower than 6.1 appears to be the c r i t i c a l point where the exudation increases (Warriss, 1982; Warriss and Brown, 1987).  However, no information on c r i t i c a l pH of prawn muscle has been  found i n the l i t e r a t u r e . 2.1.3.  Change i n ATP and related compounds  During the death struggle and a f t e r death, ATP i n f i s h muscle breaks down through the f o l l o w i n g autocatabolic pathway: ATP -» ADP -»• AMP -> IMP -»• HxR -+ Hx  9  Ehira (1976) demonstrated that inosine and hypoxanthine rates  differ  corresponding  among  species  due  to  the a c t i v i t i e s  of the  enzymes: nucleoside hydrolase and nucleoside phosphorylase,  respectively. use  fish  formation  For the same reason, Saito et a l . , (1959) suggested the  of the inosine and hypoxanthine p o r t i o n , as a r a t i o  nucleotide f r a c t i o n , to measure f i s h freshness.  o f the t o t a l  The r a t i o was expressed  as % K-value which can be c a l c u l a t e d from the f o l l o w i n g equation: HxR + Hx~ % K-value =  x 100 ATP + ADP + AMP + IMP + HxR + Hx  Among the species of f i s h tested by Ehira (1976), K-values coincided with  freshness  of the f i s h .  He also mentioned that the K-value was  influenced most strongly by the decomposition rate of IMP which was r e l a t e d to the a c t i v i t y of phosphatase i n f i s h muscle. Two recent  studies  dephosphorylation  on the degradation  of ATP showed  was not dependent on b a c t e r i a l actions while Hx forma-  t i o n was a f f e c t e d by b a c t e r i a l enzymes.  Fletcher et a l . , (1988) studied  orange roughy stored at 5°C with and without i n o c u l a t i o n with fragi.  Surette  that IMP  et  al.,  (1988) evaluated  f i l l e t s placed on i c e and held at 2-5°C.  sterile  Pseudomonas  and n o n - s t e r i l e cod  Fletcher et a l . , (1988) found  that while breakdown o f purine d e r i v a t i v e s to inosine was not a f f e c t e d by delayed  filleting,  breakdown of inosine  to hypoxanthine  b a c t e r i a l spoilage were delayed with delayed f i l l e t i n g . (1988) found that both  Pseudomonas  for the production o f i n t r a c e l l u l a r production  spp.  and Proteus  Surette et a l . , were responsible  inosine nucleosidase and hypoxanthine  was more pronounced i n f i l l e t s  Kosak and Toledo  spp.  as w e l l as  than i n gutted  (1981) found t h a t , by using  whole  fish.  c h l o r i n e pretreatment,  10  hypoxanthine accumulation i n e v i s c e r a t e d , headless, delayed.  Therefore,  scaled mullet was  as a conclusion, K-value increased  auto-degradation and then increased a c t i v i t y (Kosak and Toledo, 1981;  gradually by  r a p i d l y as a r e s u l t of b a c t e r i a l  Fletcher et a l . , 1988; Surette et a l . ,  1988). Kasemsarn et a l . , (1962) found IMP to be a f l a v o r enhancer and hypoxanthine to impart a b i t t e r t a s t e .  As i n other marine species, the  i n i t i a l l e v e l of IMP i n shrimp muscle, was considerably higher than that found i n mammalian muscle even though the i n i t i a l l e v e l o f ATP i n shrimp muscle was s i m i l a r  to that o f marine and land vertebrates  ( F l i c k and  L o v e l l , 1972). 2.1.4.  Total viable bacterial count  The majority o f n a t u r a l f l o r a i n f i s h and crustaceans are gram-negat i v e , non-fermentative b a c t e r i a (Nickelson and Finne, 1984).  The number  and types of these organisms depend on area o f catch, season, and harvest method.  The type  of spoilage  flora  will  depend  on the type o f  microorganisms that contaminate the f i s h and how the f i s h was handled. Pseudomonas  spp.,  Alteromonas  and Enterobacteriaceae are  putrefaciens,  common n a t u r a l and spoilage f l o r a i n f i s h and crustaceans.  When these  organisms reached c e r t a i n populations, t h e i r p r o t e o l y t i c a c t i v i t i e s were s u f f i c i e n t to invade underlying muscle t i s s u e of the animal.  At the l a t e  logarithmic phase o f m i c r o b i a l growth amino acids were released  due to  the p r o t e o l y s i s of muscle proteins by these b a c t e r i a l enzymes and then utilized. sulphide,  M i c r o b i a l catabolism organic  offensive smells.  sulphide,  resulted  amines,  in  formation  and ammonia  which  of hydrogen have  rather  I t was when t h i s m i c r o b i a l p r o t e o l y s i s took place that  11  spoilage  began.  Therefore, the t o t a l  population  o f these  living  b a c t e r i a , also c a l l e d t o t a l v i a b l e b a c t e r i a l count, has been used to indicate spoilage onset i n f i s h . 2.1.5.  Change i n t o t a l v o l a t i l e basic nitrogen concentration  An increase i n TVBN l e v e l i n shrimp muscle during postmortem storage on i c e has been reported by several i n v e s t i g a t o r s . produced by both n a t u r a l shrimp (Cobb and Vanderzant, 1971; Yeh muscle  V o l a t i l e bases can be  t i s s u e enzymes and b a c t e r i a l enzymes et  1978).  al.,  Amino  acids  i n shrimp  are converted to the corresponding amines by decarboxylases.  L a t e r , ammonia i s l i b e r a t e d by amine oxidase.  Ammonia was found to be  the primary component i n the TVB f r a c t i o n (Vanderzant et a l . , 1973). Production of ammonia by t i s s u e enzymes was dependent on pH and temperature (Yeh et a l . , 1978).  Two optimal pHs o f 6.0 and 8.5 and an  optimal temperature of 37°C were reported.  B a c t e r i a l decarboxylases were  a c t i v e under a c i d i c conditions while amine oxidase was able to act over a wide pH range from s l i g h t l y a c i d i c to a l k a l i n e  (Satake et a l . , 1952,  c i t e d i n Tomiyasu and Z e n i t a n i , 1957, no s p e c i f i c pH range  revealed).  Among a number of ammonia-producing enzymes tested by Yeh et a l . , (1978), only adenosine deaminase and AMP deaminase were found to be present at s i g n i f i c a n t l e v e l s i n white shrimp muscle. White  shrimp, inoculated with  Pseudomonas  species and stored f o r  11-14 days a t 5°C, had higher l e v e l s o f TVBN than the corresponding cont r o l (Cobb and Vanderzant, 1971).  Gagnon and F e l l e r s (1958) found a good  c o r r e l a t i o n between TVBN and t o t a l b a c t e r i a l count, and between TVBN and the sensory panel scores of shrimp. increased  significantly  only  after  I t was often reported that TVBN the product  had already  spoiled  12  (Jacober and Rand, 1982).  Therefore, i t d i d not r e f l e c t a l l the q u a l i t y  stages o f the prawns but rather was i n d i c a t i v e o f advanced d e t e r i o r a t i o n and spoilage. 2.1.6.  Change i n trimethylamine nitrogen concentration  Generally, TMA i s detected only i n marine f i s h and not i n freshwater f i s h (Harada, 1975). the  The presence of TMA i n f r e s h l y caught f i s h p r i o r to  onset of the b a c t e r i a l growth has been reported.  However, i n most  cases i t was found i n extremely low concentrations, normally under 1 mg TMAN/lOOgm (Hebard et a l . , 1982). Trimethylamine oxide (TMAO) i s the precursor of TMA.  TMAO i s re-  duced to TMA and to dimethylamine and formaldehyde mainly by exogenous b a c t e r i a l enzymes during spoilage of f i s h  i n c l u d i n g shrimp.  capable of reducing TMAO include most species Among these are Pseudomonas,  Bacteria  o f Enterobacteriaceae.  the n a t u r a l f l o r a of f i s h and s h e l l f i s h .  Pseudomonas  species are known to be mainly responsible f o r spoilage of  fresh  a t low temperatures which  fish  relationship  between  Alteromonas  favor  putrefaciens  their  growth.  population  A linear and TMA  production was demonstrated by Laycock and Regier (1971). Therefore, the usefulness of TMA production as an i n d i c a t o r of f i s h q u a l i t y depends on the  presence  putrefaciens,  of  TMA-producing  organisms  such  as,  Alteromonas  i n the spoilage f l o r a .  The optimum temperature f o r TMAO reduction by each microorganism i s d i f f e r e n t (Sasajima, 1973).  TMA production occurs very slowly a t c h i l l  temperatures but proceeds more r a p i d l y a t room temperature.  There i s a  lag period i n TMA formation i n f i s h stored a t low temperatures, and the lower the temperature, the longer the l a g period (Anderson and F e l l e r s ,  13  1949).  At subzero  temperatures, where the b a c t e r i a f a i l  to reach the  necessary c e l l counts to s t a r t TMAO reduction, TMA production i s almost t o t a l l y i n h i b i t e d (Sasajima, 1973, 1974). TMA upon r e a c t i o n with l i p i d i n the f i s h muscle, produces the f i s h y odour (Davies and G i l l , 1936).  The experiment on cod muscle press j u i c e  by Beatty and Gibbons (1937) revealed that TMA concentration, which was almost n e g l i g i b l e i n fresh f i s h , increased r e a d i l y a t the onset of the spoilage and c o r r e l a t e d with odours.  In c o n t r a s t , no c o r r e l a t i o n between  TMA concentrations and sensory scores (flavour and odour) was found i n roundnose grenadier (white f i s h ) (Botta and Shaw, 1976). TMA i s u s u a l l y formed under anaerobic conditions as a r e s u l t o f anaerobic r e s p i r a t i o n of b a c t e r i a which u t i l i z e s  TMAO (Watson, 1939).  This was probably because a b a c t e r i a l enzyme trimethylamine oxide reductase, which reduces TMAO to TMA, was a c t i v e only under anaerobic conditions as reported by Easter et al. (1982).  Greater TMA production was  also evident when a low oxygen permeable packaging (Murray et al., 1971).  m a t e r i a l was used  I t i s obvious that a f i l m with very low oxygen  permeability favors anaerobic growth and prevents aerobic growth. Thus the use of t h i s type of packaging f i l m may promote t h i s type of spoilage to the maximum. I t had also often been reported that TMA appeared i n s i g n i f i c a n t amounts only i n the l a t e stages o f the seafood q u a l i t y period (Jacober and Rand, 1982) .  Thus i t was rather i n d i c a t i v e of advanced d e t e r i o r a t i o n  and spoilage and d i d not r e f l e c t a l l the q u a l i t y stages during storage of seafood products.  14  2.1.7.  Changes i n concentrations of water-soluble and salt-soluble  muscle proteins Another method f o r evaluating the extent of spoilage i s to measure the concentrations of water-soluble and s a l t - s o l u b l e proteins i n muscle. According  et  to G o l l  al.,  (1977),  proteins  i n muscle  c l a s s i f i e d i n t o three groups based on t h e i r s o l u b i l i t i e s :  cells  can  be  sarcoplasmic  (proteins i n muscle plasma), stroma (connective t i s s u e ) , and m y o f i b r i l l a r (contractive  tissue) proteins.  The  sarcoplasmic  proteins are  water-  extractable.  M y o f i b r i l l a r proteins are a c t i n and myosin which form the  myofibrils.  The m y o f i b r i l l a r proteins can be extracted with high i o n i c  strength s o l u t i o n s .  The  stroma proteins comprise the connective  tissue  (collagen and e l a s t i n ) and cannot be extracted with water, a c i d , a l k a l i , or n e u t r a l s a l t s o l u t i o n s .  According to S p i n e l l i and Dassow (1982), f i s h  muscle contains approximately  20-30% of sarcoplasmic p r o t e i n s , 70-80% of  m y o f i b r i l l a r p r o t e i n s , and a n e g l i g i b l e amount of stroma p r o t e i n s . Muscle death.  protein s o l u b i l i t i e s  These changes are due  change with  time  after  an  animal's  to the e f f e c t of storage temperature on  muscle proteinases and other changes occurring i n the f i s h muscle, such as  pH  (Greaser,  1986).  Changes  in  protein  solubilities  i r r e v e r s i b l e denaturation due to postmortem pH-temperature proteolysis;  reversible  precipitation  due  to  lower  pH;  reflect:  combinations; or  altered  p r o t e i n - p r o t e i n i n t e r a c t i o n s as a r e s u l t of changes i n the concentrations of small molecular weight substrates ( f o r example, i n t e r a c t i o n of myosin and a c t i n with ATP). B a c t e r i a , which possess a very high p r o t e o l y t i c a c t i v i t y , can cause extensive breakdown of these muscle proteins (Hasegawa et al.,  Pseudomonas tragi  1970).  grew most r a p i d l y compared to the other meat spoilage  15  bacteria  (Pediococcus  tolerant  Micrococcus  (Borton  e t al.,  proteins  level  other  1970).  did not.  had greater  compared  muscle  Pseudomonas  muscle  The p o r c i n e  amounts  were  that  reported  b y Yada  showed a m a j o r  and V a n d e r z a n t , salt-soluble  spoilage  and Skura  activity  i n high  water-soluble  Similar  findings  (1981).  White  grew  for  shrimp,  among o t h e r  and coryneform  (Bacillus  experiment,  proteins there  (Cobb  was l e s s  inoculated with the fluorescent  control. most  extensive  concentration  Compared  rapidly  breakdown  of salt-soluble  to other  and t h e i r of  pronounced  muscle  proteins  meat  proteins,  and, l a t e r on,  proteins.  i s one o f t h e major  expect a p a r t i c u l a r specific  judge  caused  and water-soluble  i n water-soluble  A t the end o f t h e i r  with  Change i n colour  Colour  the  microorganisms  increase  pseudomonads  inoculated  was t h e o n l y s a m p l e ,  i n the corresponding  bacteria,  resulting  10°C  the water-soluble  muscle  controls.  p r o t e i n i n the shrimp t i s s u e  proteolytic  2.1.8.  1971).  than  Pseudomonas  a t 2°C a n d  of salt-soluble  to the corresponding  inoculated with different  bacteria),  stored  increased  tragi  i n o c u l a t e d w i t h f l u o r e s c e n t Pseudomonas, samples  and s a l t -  mesenteroides,  i n p o r c i n e muscle a t t h e l a t e r p e r i o d o f storage w h i l e t h e  tragi  proteins  Leuconostoc  on porcine  luteus)  microorganisms  Pseudomonas  beef  cerevisiae,  measurement  than  o f food  attributes  f o o d t o have a c e r t a i n c o l o u r  colour with  colour  quality  the quality  flavour quality.  or  of the food.  texture,  However,  A more m e a n i n g f u l d e s c r i p t i o n o f c o l o u r  colour  describing  i n food.  Consumers  and they a l s o Since is  associate  i t is easier  often  a colour  used  as  to a  i s n o t easy.  c a n be a c h i e v e d by e x p r e s s i n g t h e  16  colour i n 3 s c a l e s : l i g h t n e s s to darkness; hue,  such as green, yellow,  and red; and i n t e n s i t y of c o l o u r . To examine the r e l a t i o n s h i p between the colour of raw  and  canned  sockeye salmon f l e s h , Schmidt and I d l e r (1958) s u c c e s s f u l l y used a colour scale c a l l e d a value which ranges from redness to greenness,  From the  r e s u l t s , they were able to p r e d i c t the colour of canned sockeye salmon from the  colour of the raw  flesh.  Three suggested colour grades of  sockeye salmon were based on the a value. Hunter reasonably  colour uniform  measurement was estimates  developed  of perceived  as  a  colour  means  intervals  r e l a t i o n s h i p s adequately representing the colour of o b j e c t s .  to or  provide colour  Hunter co-  lour scales describe colour of an object i n 3 terms: L i s a scale of whiteness (100) to blackness (0); a i s a scale of redness (100) to greenness (-80); and b i s a scale of yellowness dimensions  of  the  Hunter  L,  a,  b,  i l l u s t r a t e d i n Figure 1 (Woyewoda et al., Red  colour  of  prawns  i s mainly  (70) to blueness (-80).  colour  pigments  can  be  caused by  light,  due  to  astaxanthin,  products.  are  the  major  Prawns d i s c o l o u r when  Oxidation or h y d r o l y s i s of these  oxygen, heat,  and  Carotenoid pigments can also be converted by lipoxygenase end  system  1986).  carotenoid pigment i n prawns ( s h e l l and f l e s h ) . the carotenoid pigments are degraded.  coordinate  The  pH  conditions. to c o l o u r l e s s  17  Figure 1.  Dimensions of the Hunter L, a, b colour coordinate system  18  2.2. Changes under carbon dioxide modified atmosphere storage Under a carbon dioxide packaging atmosphere, a decline i n the pH of a meat product occurred during the i n i t i a l Brown, 1982).  storage period (Parkin and  The higher the carbon dioxide concentration i n the input  gas, the greater the pH drop o f meat.  The pH drop occurred at the same  time as the carbon dioxide concentration i n the headspace gas i n the package decreased (Lannelongue et a l . , 1982). dioxide  absorption  Ogridziak (1986).  into  muscle  tissue  The progression of carbon  was demonstrated  by Wang and  I t was speculated that carbonic a c i d (the s o l u b i l i z e d  carbon dioxide) i n the l i q u i d phase of the treated t i s s u e may have some negative e f f e c t s on various enzymatic and biochemical pathways required for  growth  and metabolism i n microorganisms or i n the muscle t i s s u e (Da-  n i e l s et a l . , 1985).  The combined e f f e c t of these metabolic  interfer-  ences may c o n s t i t u t e a s t r e s s on the system, r e s u l t i n g i n a slowing of b a c t e r i a l growth r a t e .  Daniels et a l . ,  (1986) demonstrated that carbonic  a c i d extended s h e l f - l i f e of cod f i l l e t s at 2°C. Haines (1933) reported that carbon dioxide increased the m i c r o b i a l lag  phase and slowed growth rate of c e r t a i n microorganisms during t h e i r  logarithmic phase.  E f f e c t s of carbon dioxide modified atmosphere storage  of f i s h e r y products increased as storage temperature decreased because as temperature decreases, the s o l u b i l i t y of carbon dioxide i n an aqueous system  increases.  refrigeration  This  regulation.  i s the reason  that  Nevertheless,  in  MAP  still  requires  combination  with  r e f r i g e r a t i o n temperature, the i n h i b i t o r y e f f e c t of carbon dioxide on meat spoilage b a c t e r i a v a r i e d among b a c t e r i a l 1980).  Pseudomonas  species  (Gill  and Tan,  species grew very r a p i d l y under an a i r environment,  but were t o t a l l y i n h i b i t e d or k i l l e d under a carbon dioxide atmosphere  19  (Wang and Ogridziak, 1986). Alteromonas  outgrew of  spp.  In c o n t r a s t ,  grew slower i n modified  Pseudomonas  the retarded  spp.  Lactobacillus  spp.  atmospheres than i n a i r , but  i n modified atmosphere c o n d i t i o n s .  growth  of  and tan  Pseudomonas  spp.  under  As a r e s u l t  carbon  dioxide  environment, spoilage was delayed. While aerobic spoilage microorganisms are i n h i b i t e d by carbon dioxide at concentrations as low as 20% (Haines, 1933), carbon dioxide does not  appear to have a s i g n i f i c a n t  inhibitory  anaerobic b a c t e r i a (Huffman, 1974). anaerobic food pathogens, such as  I t i s also u n l i k e l y that f a c u l t a t i v e Staphylococcus  which grow very s l o w l y , i f at a l l , grow under a carbon dioxide  e f f e c t on the growth o f  aureus  and  Salmonella,  at r e f r i g e r a t i o n temperature, w i l l  enriched  atmosphere  temperature such as 10°C (Gray et a l . , 1983). anaerobic and aerotolerant ( G i l l , 1986).  even at an abusive  L a c t i c a c i d b a c t e r i a are  They u t i l i z e amino acids such  as v a l i n e and leucine r e s u l t i n g i n the formation of v o l a t i l e f a t t y acids which  give  cheesy  odour.  Spoilage  caused by l a c t i c  acid bacteria,  therefore, was not noticeable u n t i l long a f t e r the t o t a l v i a b l e b a c t e r i a l count had maximized. Layrisse and Matches (1984) studied changes o f  Pandalus  platyceros  stored under 50% carbon dioxide (balance a i r ) and 100% carbon d i o x i d e . They compared these changes to the changes i n prawns stored under 100% air.  From t h e i r r e p o r t , on the day of catch, the m i c r o b i a l f l o r a was  composed o f a v a r i e t y of b a c t e r i a .  S i x t y - e i g h t percent of the m i c r o b i a l  population were gram p o s i t i v e b a c t e r i a : coryneforms. Pseudomonas,  The r e s t were  Lactobacillus-like  gram negative  and Enterobacteriaciae.  bacteria:  organisms and Flavobacterium,  20  During storage under these MAP systems, gram p o s i t i v e b a c t e r i a became predominant.  I t was found that 100% carbon dioxide MAP system i n -  creased the m i c r o b i a l l a g phase to 8 days.  Layrisse and Matches (1984)  concluded t h a t , based on b a c t e r i a l counts (between l o g 1 0 6.0 to 6.5) and ammonia (approximately up to 50 mgX)  concentration,  shelf-life  of the  headless, s h e l l - o n prawns was up to 16 days f o r the MAP systems and about 14 days f o r the c o n t r o l . The c o n t r o l samples i n the study by Layrisse and Matches (1984) had a longer s h e l f - l i f e than expected because the bags (made of heat-sealable polyester/polyolefin  with  moisture  vapour  transmission  rate,  oxygen  transmission r a t e , and carbon dioxide transmission rate of 0.1 gm, 1 c c , and 27 cc/lOOin /24hr r e s p e c t i v e l y ) were sealed.  Microbial respiration  i n s i d e the bags r e s u l t e d i n the build-up of carbon d i o x i d e .  Thus, the  atmosphere i n s i d e the c o n t r o l sample bags gradually became enriched with carbon dioxide and eventually became a carbon dioxide modified atmosphere system. Nevertheless, s h e l f - l i f e of  i n another experiment by Matches and Layrisse (1985),  Pandalus  platyceros  stored at 1-2°C i n i c e under normal a i r  environment was s t i l l about 2 weeks based on sensory c r i t e r i a . The b e n e f i c i a l e f f e c t s of carbon dioxide i n c o n t r o l l i n g m i c r o b i a l growth r e s u l t i n the extended storage l i f e of shrimp (Barnett et a l . , 1978; B u l l a r d and C o l l i n s , 1978; Lannelongue et a l . , 1982; Layrisse and Matches, 1984; Matches and L a y r i s s e , 1985).  The i n h i b i t o r y e f f e c t of  carbon dioxide on m i c r o b i a l growth was due to the a c i d i c c o n d i t i o n i n duced by the d i s s o l v e d carbon dioxide i n the aqueous phase of the muscle tissue (Wang and Ogrydziak, 1986).  21  B u l l a r d and C o l l i n s (1978) reported that peeled pink shrimp stored  borealis  -1.7°C  developed  trimethylamine  i n carbon dioxide modified lower  concentrations  than those held i n i c e .  held i n carbon dioxide modified  of  refrigerated total  seawater  at  bases  and  volatile  Moreover, the colour of shrimp  r e f r i g e r a t e d seawater was  much b e t t e r  than that of shrimp held i n i c e , as i n d i c a t e d by a higher index  i n the  shrimp  stored  i n carbon  Pandalus  dioxide  modified  carotenoid refrigerated  seawater (Nelson and Barnett, 1971; B u l l a r d and C o l l i n s , 1978). Carbon dioxide modified atmosphere (80% air)  carbon d i o x i d e ,  remaining  also e f f e c t i v e l y reduced formation of amines (histamine,  tyramine,  cadavarine, and putrescine) i n whole P a c i f i c mackerel stored at  abusive  temperature of 20°C to about h a l f of that found i n mackerel stored i n a i r (Watts and Brown, 1982).  Wang and Brown (1983) found that concentrations  of ammonia, trimethylamine, and t o t a l p l a t e counts were lower i n c r a y f i s h stored under 80% carbon dioxide (balance a i r ) compared to samples stored under a normal a i r environment, a f t e r 28 days of storage. No differences i n surface colour and odour between f r e s h rock  cod  f i l l e t samples and samples stored 13 days i n the modified atmosphere were detected  by  trained  sensory  panelists,  while  both  groups  were  s i g n i f i c a n t l y d i f f e r e n t from the a i r c o n t r o l (Parkin and Brown, 1982). I t i s obvious that p l a s t i c f i l m with low d i f f u s i o n c o e f f i c i e n t s f o r gases w i l l be more appropriate to maintain a given atmosphere composition throughout the storage p e r i o d . l i f e of the f i s h products.  Packaging f i l m s also a f f e c t the  storage  As demonstrated by Debevere and Voets (1971),  the greater the oxygen permeability of the f i l m , the longer the shelfl i f e of the f i s h .  In agreement with Murray et a l . , (1971), the l a r g e r  supply of oxygen favored aerobic degradation but  i t d i d not  favor  TMA  22  production.  Nevertheless, the rate  b a c t e r i o l o g i c a l and chemical q u a l i t i e s  of d e t e r i o r a t i o n  considered from  ( t o t a l aerobic b a c t e r i a l counts,  t o t a l TMAO-reducing b a c t e r i a l counts, concentrations of TVBN and TMAN) of the pre-packed, f i s h was found to vary w i t h the type of f i s h  involved,  besides the nature of the packaging material and the storage temperature.  2.3.  Changes under nitrogen modified atmosphere storage. Unlike carbon d i o x i d e , nitrogen, which  inhibitory 1974).  effect  on the growth  i s an i n e r t gas, shows no  of aerobic microorganisms  (Huffman,  Furthermore, i t r e s u l t s i n s l i g h t l y higher anaerobic growth rate  than carbon dioxide or oxygen, and i t also does not favor the growth of l a c t i c acid bacteria.  2.4.  Multivariate analyses M u l t i v a r i a t e analysis i s applicable when one or more independent and  one or more dependent v a r i a b l e s are being considered simultaneously; each one being considered equally important at the beginning of the analysis (Massart et al., 1978). own dimension. these  Each of these measured v a r i a b l e s designates i t s  I t i s beyond the a b i l i t y of man to recognize groupings i n  multidimensional data  Multivariate  techniques  or to eliminate  can reduce  the number  excessive  information.  of dimensions  while  r e t a i n i n g the information i n a l l the data. 2.4.1.  Factor analysis  Factor a n a l y s i s i s used mainly to a i d i n i n t e r p r e t a t i o n of complex multivariate  data,  to  study  the  inter-relationships  between  the  23  variables. are  The f a c t o r analysis model assumes that the observed v a r i a b l e s  manifestations  of  a number of  unobservable  factors  (Piggott  and  Sharman, 1986).  V a r i a t i o n i n the m u l t i v a r i a t e data i s summarized i n t o  combinations  variables  of  These indices are  to  produce  called factors.  indices  The  that  are  lack of c o r r e l a t i o n means  factors are measuring d i f f e r e n t dimensions of the data. are ranked i n order.  The  uncorrelated.  f i r s t f a c t o r displays  the  the  These factors  l a r g e s t amount of  v a r i a t i o n and the second f a c t o r displays the second l a r g e s t amount of the v a r i a t i o n , and  so  on.  The v a r i a n c e s i n a f a c t o r should be as low as pos-  s i b l e , as to be n e g l i g i b l e .  In the case where the o r i g i n a l v a r i a b l e s are  very h i g h l y c o r r e l a t e d , i t i s quite conceivable that a large number of variables,  with  most  of  them measuring  similar  parameters,  can  be  adequately represented by 2 or 3 f a c t o r s .  2.4.2.  Stepwise discriminant analysis  Stepwise discriminant analysis i s a s t a t i s t i c a l  technique applied to  seek out subsets of v a r i a b l e s most useful f o r d i s c r i m i n a t i n g between the treatment groups (Powers and Ware, 1986). v a r i a b l e s , one separates the  step at a time.  variable  discrimination  f i r s t selected v a r i a b l e  samples i n t o t h e i r predetermined categories,  best d i s c r i m i n a t i o n power. selected  The  The process involves s e l e c t i n g  the  also  The  taken  most, and  into  consideration,  on.  The  process  predetermined l e v e l of s i g n i f i c a n c e regarding the  will  i n order  to  find  the first  improve  the  continues u n t i l separation  achieved, or a maximum number of steps have been taken.  variables  or has  next selected v a r i a b l e , with the  so  i s e s p e c i a l l y u s e f u l when one  optimally  has  a  been  This technique  needs to screen a large set of response a  smaller  subset that  will  provide  good  24 d i s c r i m i n a t i n g power.  I t i s also u s e f u l i n p r e d i c t i n g the c l a s s to which  unknown samples belong. The a n a l y s i s presents the r e s u l t s i n % c o r r e c t c l a s s i f i c a t i o n as i n classification  matrix  and  i n Jackknife  classification.  Unlike  the  c l a s s i f i c a t i o n matrix, Jackknife c l a s s i f i c a t i o n a l l o c a t e s each i n d i v i d u a l to i t s c l o s e s t group without using that i n d i v i d u a l to help determine the group center  (mean).  I t i s expected that an observation i s c l o s e s t to  the center of the group where the observation helps group center.  to determine that  Jackknife c l a s s i f i c a t i o n , therefore, eliminates the bias  i n the favour of a l l o c a t i n g the i n d i v i d u a l to the group that i t r e a l l y comes from (Powers and Ware, 1986; Manly, 1986).  MATERIALS AND METHODS  26  3. MATERIALS AND METHODS  3.1.  Prawn samples In 1988, two batches of headless s h e l l - o n prawns were purchased from  a  local  seafoods s u p p l i e r  (Murray F i s h Co., L t d . , Vancouver, B r i t i s h  Columbia) and transported i n waxed paperboard containers layered with crushed i c e to the laboratory. May (past spawning season). October  (approaching  The prawns f o r T r i a l 1 were purchased i n The prawns f o r T r i a l 2 were purchased i n  spawning season).  The  majority  of the prawns  purchased f o r T r i a l 2 had roe which had to be manually removed.  The roe,  located at the abdominal part of the prawn body, was p a r t i a l l y enveloped i n between the abdominal carapaces and swimmerets.  Removal of the roe  was c a r e f u l l y done by i n s e r t i n g a narrow spatula i n between the roe and prawn abdomen and then scraping the roe o f f .  However, by doing  this,  some swimmerets were also torn away and the carapaces were forced open exposing the abdominal t i s s u e .  A l l prawns were washed under c o l d tap  water and drained p r i o r to packing.  3.2.  Packaging material Packaging m a t e r i a l was DUPONT LP 920 (DuPont Canada L t d . ,  Ontario)  which  is  a  laminated  polyvinylalcohol-polyethylene.  plastic  film  of  Kingston,  polyethylene-  I t s properties are shown i n Table 1.  i s heat-sealable and quite impermeable to gases and water. x 23 cm were made from the LP 920 f i l m .  Two  headless s h e l l - o n prawns were packed i n each bag.  It  Bags of 13 cm  hundred grams  of the  3  Table 1. Properties* ' o f DUPONT LP 920 packaging f i l m  2  1  2  1  2  1  2  1  N.T.R.  = 0.013 cc(100 i n ) "  O.T.R.  = 0.05 cc (100 i n ) "  C.T.R.  - 0.3  cc (100 i n ) "  M.V.T.R. = 0.3  gm (100 i n ) "  1  1  1  1  1  1  1  1  day" atm"  day" atm" day" atm"  day" atm"  N.T.R. = Nitrogen Transmission Rate O.T.R. = Oxygen Transmisssion Rate C.T.R. = Carbon dioxide Transmission Rate M.V.T.R. = Moisture Vapour Transmission Rate  28  3.3.  Treatments Following the packaging o f the prawns, a i r i n the bags was evacuated  and replaced with 500 cc of carbon dioxide or nitrogen f o r the carbon dioxide modified atmosphere packaged (CMAP) samples and nitrogen modified atmosphere packaged (NMAP) samples r e s p e c t i v e l y . the c o n t r o l samples, were heat-sealed. i n the bags l e f t open to a i r .  3.4.  A l l bags, except f o r  The c o n t r o l samples were packed  A l l samples were stored a t 1°C.  Sampling plans The day o f packing was considered day zero.  I n T r i a l 1, sampling  was done every three days and storage was continued up to day 6 f o r the c o n t r o l samples and day 18 f o r the CMAP and the NMAP samples.  On each  sampling day, 3 bags o f each treatment were c o l l e c t e d f o r the determinat i o n o f headspace gas composition, exudate, TSA, and TSN. Another 9 bags of each treatment were t r a n s f e r r e d into a deep freezer (-18°C) awaiting the other t e s t s : TMAN, K-value, inner tissue pH, sensory e v a l u a t i o n s , and Hunter L, a, b v a l u e s . Sampling,  for Trial  2, was done every four days and storage was  continued up to day 28 f o r a l l samples.  The headspace gas compositions  i n the bags were evaluated on the day of packing, day 8, day 16, and day 28 which was the l a s t performed i n T r i a l  day of storage.  The f o l l o w i n g  analyses were  2: TSA, TSN, SPA, SPN, inner t i s s u e pH, exudate  volume, TMAN, TVBN, Hunter L, a, b values, water-soluble p r o t e i n s (WSP), s a l t - s o l u b l e proteins (SSP), and sensory e v a l u a t i o n s .  29  3.5.  Tests  3.5.1.  Headspace gas compositions i n the bags  Changes i n headspace gas atmosphere composition w i t h i n the CMAP and NMAP bags were evaluated. Headspace gas composition i n each of 3 bags of each treatment was evaluated on some selected sampling days by means of a gas chromatographic (GC) method. To analyse the composition of permanent gases of i n t e r e s t which i n cluded carbon d i o x i d e , oxygen, and n i t r o g e n , a GC system equipped w i t h 2 columns and a switching valve was used. GC-9A  (Shimadzu  Corp.,  Kyoto,  The GC system was a Shimadzu  Japan)  temperatures c o n t r o l l e d by a microcomputer. (Shimadzu  with  automated  programmed  A Shimadzu C-R3A Chromatopac  Corp., Kyoto, Japan) data a c q u i s i t i o n  s t a t i o n was used to  record the detector s i g n a l s and compute peak areas and gas concentration. A thermal c o n d u c t i v i t y detector (TCD) was used to detect oxygen and nitrogen detect  and a hydrogen-flame  carbon  dioxide.  ionization  The a n a l y t i c a l  detector  (FID) was used to  conditions  were: column oven  temperature, 50°C; i n j e c t o r temperature, 125°C; TCD temperature, 125°C; FID current, 60 mA; helium c a r r i e r gas flow r a t e , 25 cc/min. Sampling of the headspace gas i n the bag was done by i n s e r t i n g the needle of an a i r t i g h t glass syringe (with n u l l volume p o s i t i o n ) through a s i l i c o n e septum.  The s i l i c o n e septum was made by applying s i l i c o n e seal  (Canadian General E l e c t r i c , Toronto, Ontario) onto a Magic Tape (Scotch  Transparent  Brand, 3M Canada Inc., London, Ontario) and l e f t  before p u t t i n g the tape on to the bag.  to dry  The tape w i l l allow a good seal  with the p l a s t i c f i l m and the s i l i c o n e w i l l form a t i g h t seal a f t e r the  30  syringe needle i s removed.  The syringe was flushed a few times before  c o l l e c t i n g 0.5 cc of the headspace gas sample. The i n j e c t i o n volume was 0.5 c c .  The gas sample was f i r s t passed  through a porous polymer Porapak Q column (80/100 mesh s i z e , 6 f t ; Water Associates, Waters  Scientific  dioxide was separated.  L t d . , Missisauga, Ontario) where carbon  A f t e r carbon dioxide was detected, the valve was  (a 4 port switching v a l v e , Valco; Supelco Canada L t d . , O a k v i l l e , Ontario) switched a t an appropriate time to d i r e c t the r e s t o f the sample to the second column which was a Molecular Seive 5A (60/80 mesh s i z e , 6 f t ; Supelco Canada L t d . , O a k v i l l e , Ontario). separated.  Each run took 7 minutes.  Oxygen and nitrogen were then  Concentrations of carbon d i o x i d e ,  oxygen, and nitrogen i n the bags were computed.  Chromatograms of the  headspace gases i n the CMAP and the NMAP bags were shown i n Figures 2 and 3 respectively.  3.5.2.  Exudate Determination  Three bags from each treatment were used. thoroughly  swabbed with  90% ethanol,  A corner of the bag was  then a s e p t i c a l l y  cut to allow  exudate i n s i d e the bag to completely d r a i n out of the bag into a graduated  cylinder  recorded.  (10 ml) .  The exudate  from each bag was measured and  The bags had to be cut a s e p t i c a l l y because the samples i n  these bags were to be used l a t e r f o r m i c r o b i o l o g i c a l t e s t s . 3.5.3.  Microbiological tests  In T r i a l 1, aerobic and anaerobic psychrotrophic microorganisms i n prawns from each o f the 3 bags from each treatment of each sampling day were enumerated.  F i f t y grams of a sample i n the form of headless s h e l l -  31  CO CD IT)  U~i CO  O O  CM  O  CM  O CO  Time (minutes)  Figure 2.  Chromatogram o f the headspace gases i n the CMAP ba  00  o  CO CM  CM O  (V 00 CD  CM O C_>  in cu  Time (minutes)  igure 3.  Chromatogram of the headspace gases i n the NMAP bags  33  on prawns were t r a n s f e r r e d a s e p t i c a l l y from the package i n t o a s t e r i l i z e d blender j a r (Fisher S c i e n t i f i c , Ottawa, O n t a r i o ) .  Four hundred and f i f t y  m i l l i l i t e r s of 0.1% peptone ( D i f c o , D e t r i o t , Michigan) and 0.5% NaCl were added and the sample was blended to make the f i r s t d i l u t i o n .  Appropriate  s e r i a l decimal d i l u t i o n s of the sample were prepared using the peptone d i l u e n t (0.1% peptone, 0.5%  NaCl).  Each sample d i l u t i o n was (TSA)  then inoculated on to t r y p t i c a s e soy agar  using a S p i r a l P l a t e r ( S p i r a l System Instrument, Inc., Bethesda,  Maryland).  Duplicate plates were made.  Since  the samples o r i g i n a t e d  from c o l d sea water and were stored under r e f r i g e r a t i o n c o n d i t i o n s , 4°C was  selected as the incubation temperature.  The anaerobic c o n d i t i o n was  created by introducing a slow stream (15 ml/min) of nitrogen gas, humidified by passing  the gas stream through an aqueous s o l u t i o n of 5%  ascorbic a c i d and 0.1% stream.  pre-  r e z a s u r i n to scrub oxygen from the nitrogen  gas  This anaerobic chamber was made of p l e x i g l a s s with the design as  i l l u s t r a t e d i n Figure 4.  The aerobic and anerobic plates were incubated  at 4°C f o r 5 days and 7 days r e s p e c t i v e l y .  A f t e r the incubation p e r i o d ,  the number of colonies formed on each plate was counted and recorded. In  Trial  specifically  2,  additional microbiological  Sumner and  aerobically  were  to monitor the development i n the population  producing b a c t e r i a i n a l l treatments. by  tests  and  Gorczyca (1984).  The  anaerobically at 4°C  nitrogen stream was  The medium used was  carried of as  out  sulfidedescribed  inoculated p l a t e s were incubated f o r 10  days.  In T r i a l  2,  the  increased to 170 ml/min with the a d d i t i o n of a carbon  dioxide stream of 30 ml/min to further exclude the oxygen.  34  r Out  Plexiglass Anerobic  Flow meter Nitrogen gas from gas tank ST > = t \  Chamber  In  Regulator  0.1% Rezasurin 5.0% Ascorbic acid  Figure 4.  P l e x i g l a s s anaerobic chamber (38.1 cm x 33.0 cm x 50.8 cm)  35  3.5.4. Determination of pH of prawn inner tissue Each prawn was  cut lengthwise into h a l v e s .  The pH of the inner  prawn t i s s u e was measured by using a f l a t t i p pH probe (Accumet pH Meter Model 620, Fisher S c i e n t i f i c , Ottawa, Ontario).  The procedure  involved  one measurement per prawn and f i v e prawns per bag. 3.5.5. K-value determination The K-value of each sample was evaluated based on the high performance l i q u i d chromatographic (HPLC) method of Ryder (1985). 3.5.5.1.  Sample preparation  From each prawn sample, 25 grams of prawn muscle was blended i n 125 ml of c h i l l e d 0.6 M p e r c h l o r i c a c i d (BDH Chemical Co., Toronto, Ontario) s o l u t i o n f o r 60 seconds using an Osterizer Appliance  Service  (3000xg,  10  min,  Co.,  Vancouver, B.C.),  0°C)  in  a  Sorvall  (Model Galaxie 8, Sunbeam  following RC2-B  by  centrifugation  automatic  superspeed  r e f r i g e r a t e d centrifuge (Ivan S o r v a l l Inc., Newtown, Connecticut). Ten  milliters  of  the  supernatant was  collected  and  n e u t r a l i z e d to pH 6.5 to 6.8 with 1.0 M potassium hydroxide. extract  was  left  at  0°C f o r 30  minutes,  the  potassium  immediately A f t e r the perchlorate  p r e c i p i t a t e was removed by f i l t e r i n g the e x t r a c t through a 0.45 /xm type HA M i l l i p o r e f i l t e r  (Millipore®, M i l l i p o r e L t d . , Mississauga, O n t a r i o ) .  The c l e a r f i l t r a t e was  d i l u t e d to 20 ml and stored at -18°C f o r l a t e r  analysis. All  s o l u t i o n s used  i n t h i s HPLC analysis were prepared by using  deionized, d i s t i l l e d water and were f i l t e r e d through a 0.45 M i l l i p o r e f i l t e r p r i o r to i n j e c t i n g onto the column.  fxm type HA  36  3.5.5.2.  HPLC conditions  Chromatography was  performed on a Spectra-Physics SP 8700 solvent  d e l i v e r y system connected to an SP 8400 v a r i a b l e wavelength detector, and an SP 4100 computing i n t e g r a t o r (Spectra-Physics, Santa C l a r a , CA) . detector was set at 254  nm.  A reverse phase column of Supercosil LC-18 packed  with  5  The  /im s p h e r i c a l  O a k v i l l e , Ontario) was  silica  particles  (4.6 mm  ID x 25  cm)  (Supelco Canada L t d . ,  used f o r the chromatographic a n a l y s i s .  Column  temperature was maintained at 30°C with a C o n t r o l l e r Model LC-22 and a Column Heater Model LC-23 ( B i o a n a l y t i c a l System Inc., West Lafayette, Indiana). A  10  sample  loop was  used  on  the  injector.  An  MPLC  Spheri-10 RP-GU guard column (Brownlee Labs Inc., Santa C l a r a , CA) placed before the Supercosil LC-18 column.  RP-8 was  This guard column (4.6 mm ID  x 3 cm) was packed w i t h t o t a l l y porous 10 ^m Spheri-10. The guard column was changed o f t e n . An SP 4100 computing integrator was used to c a l c u l a t e i n d i v i d u a l and t o t a l peak areas. A chart speed of 1 cm/minute, an attenuation of 4 with a range of 0.04, a d e f a u l t peak width value of 6, and a d e f a u l t peak threshold value of 12 were used throughout the study. The mobile phase was a b u f f e r s o l u t i o n of 0.04 M potassium dihydrogen orthophosphate and 0.06 M dipotassium hydrogen orthophosphate.  Both  potassium dihydrogen orthophosphate and dipotassium hydrogen  phosphate  were of HPLC grade (BDH Chemical Co., Toronto, O n t a r i o ) .  isocratic  mobile phase was run at a flow rate of 2 ml/minute. f i l t e r e d through a 0.45  type HA M i l l i p o r e f i l t e r  An  The mobile phase was (Miilipore®, M i l l i -  pore Ltd., Mississauga, Ontario) before use and was prepared d a i l y .  37  Water and methanol were used to clean the column.  The mobile phase,  water, and methanol were degassed f o r 15 min p r i o r to use.  During the  e l u t i o n or the cleaning process, solutions were maintained i n a degassed state with a slow stream of helium gas. 3.5.5.3.  Reproducibility of the HPLC analysis  The r e p r o d u c i b i l i t y of the HPLC analysis was examined with a standard  solution  diphosphate  consisting  of adenosine  triphosphate  (ATP),  adenosine  (ADP), adenosine monophosphate (AMP), inosine monophosphate  (IMP), inosine (HxR), 5-bromouracil (the i n t e r n a l standard used f o r the q u a n t i f i c a t i o n of the K-value), and hypoxanthine Company, St.Louis, M i s s o u r i ) .  (Hx) (Sigma®  Chemical  The standard s o l u t i o n was i n j e c t e d onto  the column 3 times a day f o r 5 days.  A chromatogram of the nucleotide  standard working s o l u t i o n i s i l l u s t r a t e d i n Figure 5.  The r e s u l t s are  shown i n Table 2.  3.5.5.4.  Internal standard  5-Bromouracil was chosen to be the i n t e r n a l standard i n order to quantify  the K-value.  Chromatogram  of  the i n t e r n a l  standard  5-  bromouracil stock s o l u t i o n i s i l l u s t r a t e d i n Figure 6. The 5-bromouracil had a r e t e n t i o n another retention  tiny  time of 8 minutes.  peak with  a retention  time f o r the ATP.  However, i t was found to produce time  of 3.4 minutes,  the same  The minute 8 peak showed a s i g n i f i c a n t l y  greater area count than the minute 3.4 peak which seemed to be e i t h e r a degraded component or some i m p u r i t i e s . E f f e c t of t h i s t i n y peak on the q u a n t i f i c a t i o n of the K-value was investigated. tions were used.  Three d i f f e r e n t solu-  Time (minutes)  Figure 5. Chromatogram of the nucleotide standard working solut  39  Table 2. The r e p r o d u c i b i l i t y of the HPLC column. Retention Time (minutes) IMP  ATP  ADP  AMP  Hx  I.S.  HxR  Dayl  2..43  3..31  3..69  4..24  4..96  8..06  17..32  Day2  2 .61  3..69  4..12  4..72  5..56  9..05  19,.61  Day3  2..55  3..53  3..95  4.,53  5..33  8..71  18,.34  Day4  2 .58  3..58  4..00  4..60  5..43  8..87  18..80  Day5  2..55  3..51  3..93  4..53  5..35  8..72  18..41  Mean  2..54  3..52  3..94  4..52  5,.33  8..68  18..50  s.d.  0..06  0.,12  0..14  0.,16  0..20  0..33  0..74  c. v.  2,.41  3..52  3..57  3..49  3,.76  3..85  4..01  I.S.  HxR  Area count x 1000 IMP  ATP  ADP  AMP  Hx  Dayl  78..46  120.,98  98..83  142..67  192.,69  21..63  199.,22  Day2  79..58  127..19  102..02  148..98  201..50  22..34  206.,95  Day3  79,.08  125..23  102..11  147,.23  201..38  22..47  205..90  Day4  80..35  127..17  103..84  149,.45  204..14  22..47  207..99  Day5  81..37  126..30  103..82  149,.43  203..96  22,.52  207..54  Mean  79..77  125..37  102..12  147,.55  200..73  22..29  205,.52  s.d.  1..01  2..31  1..83  2..57  4..19  0..33  3..23  ; c. v  1..27  1..84  1..79  1 .74  2..09  1..50  1..57  Figure 6 . Chromatogram of the i n t e r n a l standard 5-bromouracil stock solution  41  S o l u t i o n A consisted of only the standard ATP, ADP, AMP, IMP, Hx, and HxR). solution  (3 ml) with  s o l u t i o n (a mixture of  S o l u t i o n B consisted of the standard  the a d d i t i o n of 100 fil 5-bromouracil  (Sigma®  Chemical Company, S t . L o u i s , M i s s o u r i ) . S o l u t i o n C was made by adding 100  fil 5-bromouracil  to 3 ml b u f f e r s o l u t i o n ( e l u t i n g medium).  The r e s u l t s  (Table 3) showed that there was no s i g n i f i c a n t d i f f e r e n c e i n the K-values between s o l u t i o n B and s o l u t i o n s A and C. Q u a n t i f i c a t i o n of the K-value by t h i s HPLC method was based on the stock  internal  standard  method.  bromouracil was made (46 mg/50 ml).  The o r i g i n a l  concentration  o f 5-  In each sample e x t r a c t or standard  s o l u t i o n of 2 ml, 100 fil of t h i s stock s o l u t i o n o f the i n t e r n a l standard was added.  This would give the f i n a l concentration o f the 12 nmole o f  i n t e r n a l standard i n a 10 fil i n j e c t i o n . By knowing the weight o f each component i n the standard  solution,  the concentration o f the stock i n t e r n a l standard, the d i l u t i o n , and the area counts o f a l l components involved, the concentration of each component i n the standard s o l u t i o n was determined.  With t h i s information the  K-value was c a l c u l a t e d . 3.5.5.5.  Reproducibility of the extraction  Three e x t r a c t s were made from one prawn sample. then analysed f o r the K-values. 3.5.6.  The e x t r a c t s were  The r e s u l t s are shown i n Table 4.  Trimethylamine-nitrogen determination  Trimethylamine-nitrogen  (TMAN) l e v e l i n each sample was evaluated  with the c o l o r i m e t r i c method of A.O.A.C. 1984 (sections 18.031-18.033).  Table 3.  The K values of the nucleotide standard s o l u t i o n with and without the area count of the small unknown peak (n=3)  Area Count Solution A  Solution B  IMP  51755..67  51369..67  ATP  79538..33  78628,.67  ADP  62171..67  60368..00  60368..00  AMP  104370..67  102337 .00  102337..00  Hx  59393..00  57368,.67  57368..67  (24722..67)  23609,.67  56428..67  55336,.00  55336..00  33..87  33,.64  33..74  I.S. HxR  % K value  Soluiton C  S o l u t i o n B-C  51369..67 *1298..00  24722..67  (77330..67)  23609..67  Mean  33.75  Standard d e v i a t i o n  0.09  C o e f f i c i e n t o f variance %  0.28  * •= The small unknown peak S o l u t i o n A = The nucleotide standard s o l u t i o n pluses the area count of the i n t e r n a l standard from s o l u t i o n C S o l u t i o n B = The nucleotide standard s o l u t i o n with the i n t e r n a l standard S o l u t i o n C = The i n t e r n a l standard i n the buffer s o l u t i o n (mobile phase) S o l u t i o n B-C = The area count of the small unknown peak from s o l u t i o n C was deducted from the area count o f ATP i n s o l u t i o n B  Table 4.  The r e p r o d u c i b i l i t y of the extraction  f o r K value  determination  Area Count  Extractl  Extract2  Extract3  Mean  s. d.  IMP  73983..33  79452..67  77017,.50  76817..83  2237..31  2..91  ATP  0..00  0..00  0..00  0..00  0..00  0..00  ADP  9075..67  9680..33  9267,.00  9341..00  252..34  2..70  AMP  41034..67  48734,.00  38439 .00  42735..89  4371..68  10..23  Hx  21575..33  22455..33  24265,.00  22765 .22  1119..70  4..92  I.S.  21781..33  21429,.33  21343,.00  21517,.89  189..59  0..88  HxR  39600.,00  41320..00  40023,.50  40314..50  731..72  1..82  37..95  36..75  38..76  37,.82  0..83  2..18  % K value  % c.. v.  44  One hundred grams of prawn muscle were blended f o r 1 min i n 200 ml 7.5%  trichloroacetic  acid  with  a Waring  blender  (Fisher  Scientific,  Ottawa, Ontario) which was connected to a v a r i a b l e autotransformer (Staco,  Inc., Dayton, Ohio;  0-120/140, Amp.  type 2PF  1010,  input  120  KVA  1.4,  output  10, Freq. 50/60) monitored at 80 on the s c a l e .  The  macerate was centrifuged (3000xg, 10 minutes, 0°C) i n a S o r v a l l RC2-B automatic superspeed r e f r i g e r a t e d centrifuge (Ivan S o r v a l l Inc., Newtown, Connecticut).  The  supernatants were kept at -18°C p r i o r  to  further  analyses. 3.5.7.  Total v o l a t i l e basic nitrogen determination  The steam d i s t i l l a t i o n method of Malle and Tao (1987) was used to determine l e v e l s of TVBN i n the samples. One hundred grams of prawn muscle were homogenized f o r 1 min i n 200 ml 7.5% t r i c h l o r o a c e t i c a c i d with a Waring blender (Fisher Ottawa,  Ontario)  which  was  connected  to  a  v a r i a b l e autotransformer  (Staco, Inc., Dayton, Ohio; type 2PF 1010, input 120 KVA 0-120/140, Amp.  Scientific,  1.4, output  10, Freq. 50/60) monitored at 80 on the s c a l e .  The  homogenate was centrifuged (4000xg, 5 minutes, 0°C) i n a S o r v a l l RC2-B automatic superspeed r e f r i g e r a t e d centrifuge (Ivan S o r v a l l I n c . , Newtown, Connecticut).  The  supernatant was  using a Whatman No.3 f i l t e r paper.  filtered  through a Buchner  funnel  The supernatants were kept at -18°C  awaiting f u r t h e r analyses.  3.5.8.  Sensory evaluations  A group of 7 p a n e l i s t s were a l l trained to d i f f e r e n t i a t e the following  q u a l i t i e s : odour and colour of raw prawn meat, and c o l o u r , odour,  45  f l a v o u r , and texture of cooked prawn meat. sions i n 5 consecutive weeks.  There were 5 t r a i n i n g ses-  The panel was t r a i n e d with f r e s h prawns  and prawns that were kept at r e f r i g e r a t i o n temperature to the point of spoilage.  I n each s e s s i o n , changes i n the c h a r a c t e r i s t i c s  of fresh  prawns and r e f r i g e r a t e d prawns i n both raw and cooked forms were compared and discussed.  A l l changes i n the c h a r a c t e r i s t i c s of prawns used as a  guide i n t h i s study were reported by Varga et a l . , (1972) as i l l u s t r a t e d i n Table 5. Sensory evaluation was done by using a 9-point hedonic scale: 1 = d i s l i k e extremely to 9 = l i k e extremely as shown i n Figures 7 to 9.  A  score of 9 i n d i c a t e s e x c e l l e n t q u a l i t y prawns which i s pink or orangepink body; f r e s h seaweedy odour; sweet and r i c h f l a v o u r ; f i r m and e l a s t i c texture. It  A score of 5 i s assigned the lower l i m i t of acceptable q u a l i t y .  describes  prawns with  faded  colour  and yellowish-green  staining;  s l i g h t l y musty, s l i g h t l y s t a l e , and/or very s l i g h t l y ammoniacal odour; s t a l e , sweet, and/or s l i g h t l y b i t t e r limp  texture.  quality.  a f t e r t a s t e ; and s o f t , mushy, and  Scores below 5 were considered to be of unacceptable  The panel was i n s t r u c t e d that i f any sample was d o u b t f u l , they  had a choice of not t a s t i n g that sample and to taste that sample was a voluntary a c t i o n . In T r i a l 1, seven p a n e l i s t s p a r t i c i p a t e d in, the sensory evaluation i  of  prawn samples.  available. All peeled.  In T r i a l  2, s i x out of the seven p a n e l i s t s were  Samples were presented to the panel i n random order.  prawn samples used i n the sensory evaluation were  manually  For the evaluation of odour, f l a v o u r , and texture of the prawn  samples, two prawns were put i n a aluminum f o i l cup and t i g h t l y covered with an aluminum f o i l sheet.  These cups were placed on a baking tray and  Table 5. Characteristic changes of pink prawns during storage  A p p e a r a n c e 10  Odour  F I a v o u r  T e x t u r e  Pink, Orange-pink body  Fresh, characteristic odour, seaweedy  Full fresh flavour, sweet, very rich, characteristic of fresh shrimp  Firm, elastic  9  Pale pink, slightly discolouration on heads  Fresh, seaweedy  Slight loss of flavour  Fi rm, elastic  a  Slightly faded pigment, greenish-yellow liver staining  Slightly seaweedy  Sweetish, lingering sweet aftertaste  Firm, slight loss of elasticity  7  Faded pigment, brownish discolouration on heads, greenish-yellow liver staining  Neutral to very slightly stale  Slightly sweetish to neutral  Firm, slightly elastic, some limpness  6  Faded pigment, brown discolouration on heads, yellowish-green liver staining  slightly state, fishy  Neutral to slightly stale, sweetish  Slight softness, loss of elasticity  5  Faded pigment, brown discolouration on heads, yellowish-green liver staining  Slightly musty, siightly stale, very slightly ammonical  Stale, sweet, slightly bitter aftertaste  Soft, mushy, limp  4  Very faded pigment, yellowish-green liver stafning, black discolouration on heads  Strongly ammonical, stale, musty  Stale, sweet, bitter, bitter aftertaste  Mushy, limp  Very faded pigment, very badly discoloured, very badly liver stained  Strongly ammonical, putrid  Very bitter, objectionable, putrid  Mushy  3 to 0  48  Figure 7. Sensory e v a l u a t i o n score sheet f o r cooked prawn meat odour, f l a v o u r , and texture scores  NAME  DATE  Please evaluate the COOKED prawn samples by checking your r a t i n g s on t h e i r odour, f l a v o r , and texture. Commenting on each parameter i s encouraged. Sample code Odour Flavor Like extremely Like very much Like moderately Like s l i g h t l y Neither l i k e nor d i s l i k e Dislike slightly D i s l i k e moderately D i s l i k e very much D i s l i k e extremely  ,  Sample code Odour Flavor Like extremely L i k e very much Like moderately Like s l i g h t l y Neither l i k e nor d i s l i k e Dislike slightly D i s l i k e moderately D i s l i k e very much D i s l i k e extremely  Texture ,  _____  Sample code Odour Flavor Like extremely Like very much Like moderately Like s l i g h t l y • Neither l i k e nor d i s l i k e Dislike slightly D i s l i k e moderately D i s l i k e very much D i s l i k e extremely  Texture  ,  Texture j  1  | Comments : | | | | | | | | | | | Comments : | | | | | | | | | | | Comments : | | | | | | | | | |  50  Figure 8. score  Sensory e v a l u a t i o n score sheet f o r cooked prawn meat colour  51  NAME  DATE  Please evaluate the COOKED prawn samples by checking your r a t i n g s on t h e i r c o l o u r . Commenting on each parameter i s encouraged. Sample code  Sample Code  Sample code  Sample Code  Like extremely Like very much Like moderately Like s l i g h t l y Neither l i k e nor d i s l i k e Dislike slightly D i s l i k e moderately D i s l i k e very much D i s l i k e extremely  Like extremely Like very much Like moderately Like s l i g h t l y Neither l i k e nor d i s l i k e Dislike slightly D i s l i k e moderately D i s l i k e very much D i s l i k e extremely  " •  Sample code Like extremely Like very much Like moderately Like s l i g h t l y Neither l i k e nor d i s l i k e Dislike slightly D i s l i k e moderately D i s l i k e very much D i s l i k e extremely Comments:  Sample Code  _  j  52  Figure 9. Sensory e v a l u a t i o n score sheet f o r raw prawn meat odour and colour scores  NAME  DATE  Please evaluate the RAW prawn samples by checking your r a t i n g s on t h e i r colour and odour. Commenting on each parameter i s encouraged. Sample code Colour Odour Like extremely Like very much L i k e moderately Like s l i g h t l y Neither l i k e nor d i s l i k e Dislike slightly D i s l i k e moderately D i s l i k e very much D i s l i k e extremely  Sample code Colour Odour -  -  Sample code Colour Odour  Sample code Colour Odour  Sample code Colour Odour  Sample code Colour Odour  Like extremely Like very much Like moderately Like s l i g h t l y Neither l i k e nor d i s l i k e Dislike slightly D i s l i k e moderately D i s l i k e very much D i s l i k e extremely  Like extremely Like very much Like moderately Like s l i g h t l y Neither l i k e nor d i s l i k e Dislike slightly D i s l i k e moderately D i s l i k e very much D i s l i k e extremely  Comments:  54  put  i n a conventional oven preheated at 200°C. Cooking was continued f o r  25 min. For put  evaluation of the colour of the prawn samples, two prawns were  i n a c l e a r glass bowl and then t i g h t l y covered with an aluminum  sheet. out  Cooked samples were kept warm at 60°C f o r s e r v i n g .  These bowls were placed on a baking t r a y .  foil  Cooking was c a r r i e d  i n the same manner as above. Figure  10 showed the temperature gradient' a t the center of the  preheated (200°C) conventional oven a f t e r i t was opened and then closed (simulating that samples were being loaded).  The temperature gradient  i n s i d e the edible prawn t i s s u e during cooking i s shown i n Figure 11. I t was shown that w i t h i n 15 min a f t e r loading, the temperature i n s i d e the edible prawn t i s s u e was maintained at approximately 100°C. For put  the odour and colour of the raw prawn samples, two prawns were  i n a c l e a r glass bowl and then t i g h t l y covered w i t h an aluminum  sheet.  These bowls were kept i n a 4°C cold room p r i o r  foil  to the panel  tests. For der  each t e s t , samples were presented to each p a n e l i s t i n random or-  to balance out any order e f f e c t s  that might occur.  The prepared  samples were placed on a white paper plate before serving to each panelist. prawn  F i r s t , the evaluations of odour, f l a v o u r , and texture of the cooked samples  were  done  under  the red-masked, l i g h t .  Second, the  •I  evaluation  of colour  of the cooked  prawn  samples;  and t h i r d , the  evaluation of odour and colour of raw prawn samples were c a r r i e d out under normal white l i g h t . taste  and unsalted  Room temperature water with a s l i g h t  lemon  soda crackers were served so each p a n e l i s t  could  cleanse t h e i r mouth between samples.  55  205  0  5  10  15  20  25  30  Minutes after loading  Figure 10. Temperature gradient at the center of the preheated (200°C) conventional oven  Figure 11. Temperature gradient inside the edible prawn tissue during cooking at 200°C  57  3.5.9. Hunter L, a, b values The colours of raw, headless, s h e l l - o n prawns were measured by an o b j e c t i v e method based on the Hunter opponent-colour s c a l e s : L, a, b values f o r lightness/darkness, redness/greenness, and scales  respectively.  Measurement  was  performed  yellowness/blueness using  a  spectrocolorimeter (Labscan I I IBM 0°/45°, Hunter Associates Inc., Reston, V i r g i n i a ) . disposable  plastic  reading  Laboratory,  A s i n g l e prawn was placed on a new s t e r i l i z e d  petridish  (Fisherbrand,  S c i e n t i f i c Company, Ottawa, O n t a r i o ) . single  Hunterlab  100x15mm Standard,  Fisher  An aperture s i z e of 1.0 cm with a  per prawn was used throughout the study.  The Hunter  opponent-colour values were determined on the same day as the sensory evaluation t e s t s were conducted. 3.5.10. Water-soluble and salt-soluble protein determinations Water-soluble  and s a l t - s o l u b l e proteins were extracted according to  the procedure used by Cobb and Vanderzant (1971).  Water-soluble  proteins  were extracted from 25 gm prawn t i s s u e using 50 ml d i s t i l l e d water.  To  extract s a l t - s o l u b l e p r o t e i n s , the residue was blended with 50 ml of pH 7.2 b u f f e r which consisted of 0.45 M KC1, 0.0157 M Na2HP04, and 0.0031 M KH2P04. The p r o t e i n n i t r o g e n l e v e l s of the extracts were determined using BCA P r o t e i n Assay Reagents (Pierce Chemical Company, Rockford,  Illinois).  Incubation at 60°C f o r 30 minutes was used throughout the study. 3.6.  S t a t i s t i c a l analyses Factor a n a l y s i s and stepwise discriminant a n a l y s i s were performed on  an Amdahl 470 V/8.  Both analyses are i n the BMDP (Biomedical Programs,  58  BMDP S t a t i s t i c a l  Software  Inc., 1985).  Factor a n a l y s i s  and stepwise  discriminant analysis were performed on selected v a r i a b l e s from the data of T r i a l 2 (n=720) .  The data input i s as shown i n Figure 12 and was  c a l l e d "multivariate data".  The m u l t i v a r i a t e data included the data of  20 v a r i a b l e s obtained from each of 3 bags of each treatment (the c o n t r o l , the  CMAP, and the NMAP assigned  as treatment  number  1, 2, and 3  r e s p e c t i v e l y ) during the 12 day storage p e r i o d (4 sampling days).  Only  the data obtained up to day 12 were analysed because by day 12, prawns from a l l treatments were of unacceptable q u a l i t y based on sensory point of view ( o v e r a l l sensory s c o r e s ) .  The data was rearranged i n such a way  that time function could be included i n the analyses with a l l required data f o r each v a r i a b l e presented as one data subset. For f a c t o r a n a l y s i s , program P4M i n the BMDP was applied to the m u l t i v a r i a t e data. so  that  D i r e c t quartimin r o t a t i o n was used to rotate factors  the variance  variables.  o f the squared  loadings was maximized  within  This r o t a t i o n method s i m p l i f i e s v a r i a b l e s by producing one or  more large loadings and the r e s t as near to zero as p o s s i b l e . For stepwise d i s c r i m i n a n t a n a l y s i s , program P7M i n the BMDP programs was  applied to the m u l t i v a r i a t e data.  In t h i s study, the purpose of  using the stepwise d i s c r i m i n a n t analysis was to o b t a i n equations that a i d the c l a s s i f i c a t i o n o f these prawn products according to t h e i r degree of freshness  or s p o i l a g e .  TMAN  concentration was  selected  as the  discriminant v a r i a b l e because i t i s the most s u i t a b l e freshness/spoilage i n d i c a t o r f o r the prawns i n t h i s study. spoilage  compounds were  the r e s u l t s  This was because while other  of b a c t e r i a l  and t i s s u e  enzyme  a c t i v i t i e s , TMA was mainly produced by b a c t e r i a during storage and i t s  Figure 12.  M u l t i v a r i a t e data input format  MAP Bag Day Drip pH 0 1 1 0.0 7.40 1 0 2 0.0 7.60 1 3 0 0.0 7.34 2 0 1 0.0 7.40 2 2 0 0.0 7.60 2 3 0 0.0 7.34 3 0 1 0.0 7.40 3 0 2 0.0 7.60 3 3 0 0.0 7.34 1 1 4 8.0 7.31 1 4 2 5.6 7.33 4 1 3 5.5 7.23 2 4 1 7.0 6.50 4 11.0 6.41 2 2 4 2 3 6.5 6.44 3 1 4 7.7 7.13 3 4 2 7.8 7.10 3 3 4 5.2 7.06 1 1 8 4.9 7.89 1 2 8 4.2 7.65 3.7 7.74 1 8 3 9.2 6.64 2 8 1 2 2 3 3 3 1 1 1 2 2 2 3 3 3  2 3 1 2 3 1 2 3 1 2 3 1 2 3  8 8 8 8 8 12 12 12 12 12 12 12 12 12  2.4 6.1 3.8 4.5  5.36  TMAN TVBN 0.06 0.38 0.07 0.55 0.04 0.61 0.06 0.38 0.07 0.55 0.04 0.61 0.06 0.38 0.07 0.55 0.04 0.61 3.06 1.46 2.83 1.25 1.61 1.17  WSP 30.17 26.79 26.27 30.17 26.79 26.27 30.17 26.79 26.27 38.33 39.74 36.68  SSP 34.10 43.86 39.68 34.10 43.86 39.68 34.10 43.86 39.68 41.65 47.27 45.73  37.28 37.89 37.07 37.28 37.89 37.07 37.28 37.89 37.07 38.32 37.18 41.18  L a b 12.04 12.37 12.52 11.88 11.66 11.83 12.04 12.37 12.52 11.88 11.66 11.83 12.04 12.37 12.52 11.88 11.66 11.83 10.73 11.68 10.95 11.56 13.29 12.96  5.43  2.08  1.40  2.90  1.08  29.45  55.99 40.54  13.17 12.60  6.03 6.09 6.12 6.15 7.58 7.66 7.59 7.63 7.17 7.21  2.21 2.99 4.27 4.21 4.13  2.68 3.07 4.47 4.27 4.07  2.24 1.20 4.08 3.66 2.71  1.07 0.88 1.17 1.06 1.15  34.00 31.09 41.04 42.44 48.36  53.47 54.41 52.70 54.34 46.73  38.17 37.11 38.95 38.00 39.71  12.77 11.95 14.16 14.21 13.45  11.58 12.10 11.65 12.21 13.38  8.48 8.58 8.58 8.64 8.47 8.54 6.46 7.05  5.10 4.97 4.89 3.28  4.00 4.10 4.18 2.99  13.60 12.00 11.90 5.85  0.96 0.69 0.58 0.37  45.87 43.14 43.31 25.39  43.71 40.41 47.07 50.65  37.97 38.12 38.28 40.73  11.99 11.32 11.07 12.96  13.39 12.78 13.30 12.74  6.17 7.22  3.32 3.34  2.10 2.95  1.90 3.30  0.24 0.53  22.15 44.34 39.20 26.02 48.20 40.63  7.39 7.94 7.94 5.12 7.40 7.76 7.89 5.08 7.31 8.01 8.07 5.12 8.04 9.95 9.92 6.60 8.12 10.06 9.93 6.98 8.11 9.83 9.82 6.72 6.69 8.03 8.04 5.33 6.67 7.9 8.03 5.12  4.68 4.94 5.12 6.30 6.92 6.51 4.54 4.70  32.05 13.80 27.15 23.76 28.38 28.93 26.51 10.34  0.91 0.63 0.78 1.02 1.21 1.17 0.72 0.42  7.14 7.46 7.47 7.51  4.65 5.15 5.45 5.05  8.91 41.25 37.07 52.80  0.55 1.31 1.00 1.39  8.7 6.49 11.9 6.57 3.2 2.3 6.4 5.8 5.6 4.9 13.1 7.8  TSA TSN SPA SPN 6.35 5.97 1.70 0.40 6.05 6.10 2.16 1.00 5.24 4.99 1.48 0.88 6.35 5.97 1.70 0.40 6.05 6.10 2.16 1.00 5.24 4.99 1.48 0.88 6.35 5.97 1.70 0.40 6.05 6.10 2.16 1.00 5.24 4.99 1.48 0.88 7.87 7.95 4.24 4.03 7.47 7.49 4.19 4.15 7.55 7.70 4.25 4.18  8.38 9.33 9.19 9.19  6.20 7.32  8.37 8.86 8.93 8.95  5.34 5.13 4.99 4.86  40.75 63.05 35.18 55.26 41.38 52.47 106.01 21.30 102.01 26.24 103.62 30.16 35.16 54.30 36.76 50.50 46.13 52.77 60.61 53.37  59.52 62.92 55.73 52.09  13.04 12.52 15.48 13.32  39.53 39.63 40.27 35.77 36.05 35.54 41.80 40.81  14.34 15.32 16.23 10.60 11.64 10.27 13.95 13.84  11.85 11.98 12.50 11.85 12.88 12.54 12.66 14.01  43.56 40.37 39.68 39.62  14.47 13.92 14.26 14.31  12.79 12.56 11.98 11.61  OVERALL 6.50 6.50 6.50 6.50 6.50 6.50 .50 .50 .50 .67 ,67 .67 .47 ,47 ,47 5.75 5.75 5.75 4.45 4.45 4.45 5.04 5.04 5.04 5.31 5.31 5.31 3.42 3.42 3.42 4.81 4.81 4.81 4.43 4.43 4.43  RC 6.33 6.33 6.33 6.33 6.33 6.33 6.33 6.33 6.33 6.17 6.17 6.17 7.17 7.17 7.17 6.00 6.00 6.00 4.50 4.50 4.50 5.83 5.83 5.83 6.00 6.00 6.00 17 17 17 83 83 83  RO 7.33 7.33 7.33 7.33 7.33 7.33 7.33 7.33 7.33 6.67 6.67 6.67 7.00 7.00 7.00 6.00 6.00 6.00 4.00 4.00 4.00 5.83 5.83 5.83 4.67 4.67 4.67 2.33 2.33 2.33 6.00 6.00 6.00 3.50 5.40 3.50 5.40 3.50 5.40  CC 6.67 6.67 6.67 6.67 6.67 6.67 6.67 6.67 6.67 5.67 5.67 5.67 4.50 4.50 4.50 6.83 6.83 6.83 5.33 5.33 5.33 4.17 4.17 4.17 6.67 6.67 6.67 3.83 3.83 3.83 4.50 4.50 4.50 6.00 6.00 6.00  CO 5.33 5.33 5.33 5.33 -5.33 5.33 5.33 5.33 5.33 4.00 4.00 4.00 3.83 3.83 3.83 4.50 4.50 4.50 2.83 2.83 2.83 4.00 4.00 4.00 4.17 4.17 4.17 2.33 2.33 2.33 3.50 3.50 3.50 2.17 2.17 2.17  CF 6.67 6.67 6.67 6.67 6.67 6.67 6.67 6.67 6.67 5.17 5.17 5.17 5.17 5.17 5.17 5.17 5.17 5.17 3.83 3.83 3.83 5.60 5.60 5.60 5.00 5.00 5.00 2.50 2.50 2.50 4.67 4.67 4.67 4.50 4.50 4.50  CT 6.67 6.67 6.67 6.67 6.67 6.67 6.67 6.67 6.67 6.33 6.33 6.33 5.17 5.17 5.17 6.00 6.00 6.00 6.20 6.20 6.20 4.80 4.80 4.80 5.33 5.33 5.33 5.33 5.33 5.33 6.33 6.33 6.33 5.00 5.00 5.00 ON  o  61  concentration  increased  gradually  throughout the 12 days storage.  in a  considerably  steady  rate  Moreover, i t s a s s o c i a t i o n with  fishy  odour and off-odours i n seafood products makes i t s u i t a b l e to be used to i n d i c a t e the q u a l i t y of seafood products.  Based on studies by C a s t e l l et  a l . , (1958) and C a s t e l l and Greenough (1958), TMAN concentration was used to  classify  fish  quality  into  three  grades.  Grade 1 f i s h  or prime  q u a l i t y f i s h contained 0 to 1 mg TMAN/100 gm muscle.  Grade 2 f i s h or  marketable f i s h contained 1 to 5 mg TMAN/100 gm muscle.  Grade 3 f i s h or  unacceptable  f i s h contained more than 5 mg TMAN/100 gm muscle.  I n some  sectors o f Japanese and A u s t r a l i a n markets, 5 mg TMAN/100 gm muscle t i s s u e i s also used as the a c c e p t a b i l i t y l i m i t f o r shrimp (Montgomery et  a l . , 1970).  In t h i s study, three ranges of TMAN concentration were used  to e s t a b l i s h outpoints f o r separation of the prawn data i n the stepwise discriminant a n a l y s i s .  These ranges were TMAN concentrations up to 1.00,  1.01 to 5.00, and more respectively.  than  5.00 mg TMAN/100  gm of prawn  tissue  These three ranges of TMAN concentration represent good,  acceptable, and unacceptable q u a l i t y r e s p e c t i v e l y . Obviously, to evaluate the q u a l i t y o f a h i g h l y perishable food product,  the t e s t s  should  be  simple,  reliable,  less  time-consuming,  inexpensive, and the number of the tests should also be minimized. achieve  this,  first,  variable pairs  that had high  correlations  selected from the c o r r e l a t i o n matrix obtained by the f a c t o r  To were  analysis.  Second, v a r i a b l e s that gave s i m i l a r information were e l i m i n a t e d .  After  the stepwise discriminant analyses were run on the data of these selected v a r i a b l e s , only the v a r i a b l e ( s ) that produced highest percent classification  o f these prawn samples were the f i n a l s e l e c t i o n .  correct  62  Analysis o f variance (ANOVA), Tukey's t e s t , student's paired T - t e s t , and Friedman two-way ANOVA were performed using SYSTAT program (Systat Inc.,  1988).  I n the SYSTAT program, by running a Tukey's t e s t , an ANOVA  was automatically included and an ANOVA table was produced. were done on data of each treatment Tukey's  test  significantly  determines different.  the groups A  These t e s t s  and data o f each sampling day. o f data  student's  that were and were not  paired  T-test  was  done on  m i c r o b i o l o g i c a l data to determine i f there was any s i g n i f i c a n t difference between aerobic and anaerobic microbial populations. Friedman two-way ANOVA was applied to sensory scores given by each panelists.  This s t a t i s t i c a l analysis ranks the sensory scores p r i o r to  proceeding w i t h the ANOVA thus overcoming the v a r i a t i o n that was l i k e l y to happen when each p a n e l i s t , even when the same measurment method was used, the scale was not quite exactly the same to a l l p a n e l i s t s . day v e r i a t i o n s were also overcome by t h i s method.  Day to  Another two-way ANOVA  (O.Mahony, 1986) was applied to the sensory data from each p a n e l i s t to determine i f there was any s i g n i f i c a n t difference among judges.  63  4. RESULTS  64  4.  4.1.  RESULTS  Trial 1  4.1.1.  Headspace gas composition  A s l i g h t increase i n carbon dioxide concentration i n the NMAP bags occurred during storage  (Figure 13).  Changes i n nitrogen concentration  i n the headspace of the bags were proportional to the changes o f the carbon dioxide concentration.  Figure 14 shows that at day 0, most but  not a l l of the a i r i n the bag was evacuated before backflushing with carbon d i o x i d e .  I t shows that carbon dioxide concentration i n the CMAP  bags dropped soon a f t e r the storage s t a r t e d and l a t e r slowly increased. Carbon dioxide concentrations (P<0.01) during  storage  significantly different  i n the CMAP bags d i f f e r e d  with  that  from the r e s t .  the NMAP bags also d i f f e r e d  of day 3 being  significantly  the lowest and  Carbon dioxide concentrations i n  significantly  (P<0.01) during storage  carbon dioxide concentration of each sampling day being different 4.1.2.  with  significantly  from the other days. Exudate formation  The highest exudate volume occurred i n CMAP prawns followed by the NMAP and the c o n t r o l prawns r e s p e c t i v e l y (Figure 15).  By day 18,  the  amount o f exudate formed i n the CMAP prawns was about twice that formed i n the NMAP prawns.  No s i g n i f i c a n t  (P>0.01) changes i n exudate volume  during 6 days storage were found i n each treatment (Table 6) . days  storage,  however, change i n the exudate volume was  During 18 significant  65  100  If  96136 90.80  80  60  C (D CL  40  20 9.04 0  0.5SQ: DayO  2Jl7( Day3  3_3  ii:  Day9  Day18  Figure 13. T r i a l 1: Concentrations of carbon d i o x i d e , oxygen, and nitrogen gas i n the NMAP bags stored at 1°C  66 r  100  80  C <U  Q_  93.11 88.09  1 i  88.30  81.01  60  40  20  6.77  1  .74 5.74  0  m  .in DayO  .Z Day3  i.1 f Day9  11.59  mi Day18  Figure 14. T r i a l 1: Concentrations of carbon d i o x i d e , oxygen, and nitrogen gas i n the CMAP bags stored at 1°C  67  0  3 . 6  9  12  15  18  Days of Storage  Figure 15. T r i a l 1: Exudate volumes of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  Table 6. I.  F - s t a t i s t i c s (a) from ANOVA on data o f T r i a l 1  Data o f 6 days storage  Variables (b)  Control  CMAP  Exudate (c) pH TSA TSN TMAN IMP ADP AMP Hx HxR K-value L a b  0.585 1.666 293.742 326.853 46.240 11.397 3.611 65.379 12.152 1.544 29.766 0.007 0.283 5.864  0 .195 35 .299 15 .696 19 .339 2 .643 3 .564 5 .543 26 .668 7 .467 0 .149 119 .973 17 .164 17 .919 13 .363  II.  Exudate pH TSA TSN TMAN IMP ADP AMP Hx HxR K-value L a b  (b) (c) (d)  ** ** ** *  ** ** ** * ** * ** ** ** **  0.567 0.499 106.344 178.23 12.569 11.733 0.736 26.596 28.812 0.754 161.374 12.659 5.314 10.711  ** ** ** ** ** ** ** ** **  Data of 18days storage  Variables  (a)  ** ** ** **  NMAP  CMAP  3 .386 18 .418 69 .798 65 .036 37 .792 11 .834 5 .498 49 .399 12 .691 1.618 71 .106 9 .055 9 .235 9 .325  NMAP * ** ** ** ** ** **  **  ** ** ** ** **  * = s i g n i f i c a n t a t 5% l e v e l ** = s i g n i f i c a n t a t 1% l e v e l See A b b r e v i a t i o n L i s t Only data of day 3-day 6; no exudate on day 0 Data became zero during storage  2.401 6.825 70.687 141.902 146.387 NV 6.238 52.140 31.381 24.635 183.839 11.231 16.285 8.667  ** ** ** ** (d)  **  ** ** ** ** ** ** **  Table 7. F - s t a t i s t i c s (a) from ANOVA on data of individual sampling day of T r i a l 1 (b)  Variables (c)  Exudate PH TSA TSN TMAN IMP ADP AMP Hx HxR K-value L a b  Variables  Exudate PH TSA TSN TMAN IMP ADP AMP Hx HxR K-value L a b  (a) (c) (b)  Day3  16.405 59.445 69.834 152.911 1.518 1.075 12.908 0.892 1.234 2.073 0.569 . 7.655 2.213 1.711  Day6 ** ** ** ** **  *  Dayl2  0.307 336.538 96.571 97.595 25.181 2.999 0.350 0.131 20.172 2.245 2.333 1.411 9.136 0.998  3.521 51.754 475.244 342.504 1.741 3.329 2.925 3.574 0.404 2.417 0.229 7.372 13.927 1.404  Day9  ** ** **  * **  Dayl5  ** ** ** **  *  **  5.647 133.225 11.521 63.034 32.107 5.117 0.508 0.164 7.329 6.922 4.505 0.186 4.571 3.228  8.204 169.615 409.862 139.754 31.087 26.370 3.357 3.019 4.384 79.849 9.071 0.637 8.269 11.158  ** ** ** ** **  ** *  Dayl8  ** * ** **  **  28.735 44.569 15.556 12.956 224.210 3.857 4.701 0.628 9.899 20.555 4.155 0.212 3.058 0.559  * = s i g n i f i c a n t at 5% l e v e l ** = s i g n i f i c a n t at 1% l e v e l See Abbreviation L i s t For day 3 and day 6, compared c o n t r o l , CMAP, and NMAP For day 9 to day 18, compared CMAP and NMAP  ** ** * * **  * *  70  (P<0.05) i n the CMAP prawns.  I t was on day 3 that d i f f e r e n c e s i n exudate  volume of prawns from these three treatments were s i g n i f i c a n t (Table 7 ) . 4.1.3.  Tissue pH  The prawns at the time of packing had a pH o f about 7.2 (Figure 16). The pH o f the c o n t r o l prawns increased during storage.  After a slight  i n i t i a l drop, the t i s s u e pH of the the NMAP prawns s l i g h t l y  increased.  A f t e r day 9, the pH o f the NMAP prawns remained i n the range o f 7.3 to 7.4 u n t i l the end of storage.  The t i s s u e pH o f the CMAP prawns dropped  to 6.6 w i t h i n 3 days of storage and then increased s l i g h t l y to 6.9 by the l a s t day (day 18) o f storage. There were no s i g n i f i c a n t  (P>0.05) changes i n t i s s u e  pH i n the  c o n t r o l and the NMAP prawns during the f i r s t 6 days of storage. contrary,  the changes  i n the t i s s u e  s i g n i f i c a n t during the f i r s t  pH  o f the CMAP  6 days of storage.  On the  prawns  were  S i g n i f i c a n t changes i n  the t i s s u e pH were found during the 18 days storage o f the CMAP and the NMAP prawns (P<0.01, Table 6 ) . three  treatments  were  The t i s s u e pH values of the prawns from  significantly  different  on each  sampling  day  throughout the 18 days storage (P<0.01, Table 7 ) .  4.1.4. Microbiology The highest t o t a l psychrotrophic b a c t e r i a l counts were found i n the c o n t r o l prawns followed by the NMAP and the CMAP prawns r e s p e c t i v e l y (Figure  17).  The i n i t i a l  counts  of t o t a l  psychrotrophic b a c t e r i a were about l o g 1 0 5.7.  aerobic  and  anaerobic  There was no s i g n i f i c a n t  difference (P>0.05) between t o t a l aerobic psychrotrophic b a c t e r i a l count (TSA) and t o t a l anaerobic psychrotrophic b a c t e r i a l count (TSN)  i n prawns  6.50'  0  3  6  9  12  15  18  Days of Storage  Figure 16. T r i a l 1: Inner t i s s u e pHs of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  72  Figure 17. T r i a l 1: T o t a l aerobic and t o t a l anaerobic psychrotrophic b a c t e r i a l counts of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C (+ = aerobic, - = anaerobic)  73  from a l l treatments  during storage.  increased most r a p i d l y storage.  TSA and TSN o f the c o n t r o l prawns  and reached about l o g 1 0  8.5 w i t h i n 6 days of  TSA and TSN of the NMAP prawns increased l e s s r a p i d l y  than  those i n the c o n t r o l prawns and gradually l e v e l l e d o f f toward the end of storage. initial  I n the CMAP prawns, however, TSA and TSN remained close to the l e v e l u n t i l day 3 before they increased gradually a t a slower  rate than those i n the NMAP prawns u n t i l the end o f storage. Changes  i n TSA and TSN during  s i g n i f i c a n t (P<0.01, Table 6 ) .  storage  of each  treatment  were  S i g n i f i c a n t d i f f e r e n c e s were also found  among treatments f o r both TSA and TSN, on every sampling day (Table 7 ) . 4.1.5.  K-values  S i m i l a r K-values were found i n a l l samples up to day 6 o f storage (Figure 18).  A f t e r day 6 to the end of the storage, the NMAP prawns had  higher K-values than the CMAP prawns.  Changes i n K-value i n each treat-  ment during 6 days storage as w e l l as 18 days storage were s i g n i f i c a n t (P<0.01, Table 6 ) .  The d i f f e r e n c e among K-values of the CMAP and NMAP  prawns was s i g n i f i c a n t only on day 9 (P<0.05, Table 7 ) . No ATP was detected i n the muscle t i s s u e of the raw m a t e r i a l prawns. ADP concentration decreased most r a p i d l y i n the CMAP prawns followed by the c o n t r o l and the NMAP prawns r e s p e c t i v e l y up to approximately (Figure  19).  remained about  A f t e r t h a t , the ADP concentration the same while  continued to decrease.  day 12  i n the CMAP prawns  ADP concentration  i n the NMAP prawns  Only the changes i n the ADP concentration o f the  CMAP prawns were s i g n i f i c a n t during 6 days storage  (P<0.05, Table 6 ) .  During 18 days storage, ADP concentration changed s i g n i f i c a n t l y i n both the CMAP and the NMAP prawns (P<0.01, Table 6 ) .  Differences among the  74  Figure 18 T r i a l 1: K-values of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  75  8  _  1' 0  3  6  9  12  15  18  Days of Storage  Figure 19. T r i a l 1: Adenosine diphosphate concentrations of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  76  ADP  concentrations  o f the prawns  from  these  three  treatments  were  s i g n i f i c a n t (P<0.01, Table 6) only on day 3. AMP concentrations i n the c o n t r o l , the CMAP, and the NMAP prawns decreased very r a p i d l y i n a s i m i l a r manner (Figure 20). A f t e r day 6, the decrease i n the AMP concentration i n the CMAP and the NMAP prawns slowed down u n t i l  the end of storage.  changed s i g n i f i c a n t l y But  there  were  concentrations  I n each treatment, AMP concentration  (P<0.01) during both storage  no s i g n i f i c a n t  of prawns  differences  from these  periods  (P>0.05)  treatments  (Table 6 ) .  among  the AMP  on each sampling day  (Table 7) throughout the storage. IMP concentration decreased r a p i d l y i n a l l samples with the most r a p i d decrease occurring i n the c o n t r o l prawns followed by the NMAP and the CMAP prawns r e s p e c t i v e l y (Figure 21). Changes i n the IMP concentration  i n the c o n t r o l and the NMAP prawns during  significant  (P<0.01, Table  6).  A  significant  6 days storage change  i n the IMP  concentration of the CMAP prawns was found during 18 days storage 6) .  were  (Table  There were no s i g n i f i c a n t differences among the IMP concentrations  of these  prawns on each sampling  day during storage  except on day 9  (Table 7 ) . Inosine concentration decreased most r a p i d l y i n the c o n t r o l prawns (Figure 22).  I n the NMAP prawns, inosine increased and then decreased  very r a p i d l y .  Inosine i n the CMAP prawns also increased, but more slowly  and f o r a longer time compared to that i n the NMAP prawns, and then decreased a t a slower rate than i n the NMAP prawns.  S i g n i f i c a n t changes  i n the inosine concentration were found only i n the NMAP prawns during 18 days storage  (P<0.01, Table 6 ) .  Inosine concentrations of these (CMAP  77  F i g u r e 20. T r i a l 1: A d e n o s i n e monophosphate c o n c e n t r a t i o n s o f prawns s t o r e d under a e r o b i c c o n t r o l , carbon d i o x i d e , and n i t r o g e n atmospheres a t 1°C  78  60  Days of Storage  Figure 21. Trial 1: Inosine monophosphate concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C  79  Figure 22. T r i a l 1: Inosine concentrations of prawns stored under aerobic c o n t r o l , carbon dioxide, and nitrogen atmospheres at 1°C  80  and NMAP) prawns were s i g n i f i c a n t l y d i f f e r e n t only on day 9 and day 18 (Table 7 ) . Hypoxanthine developed gradually at a s i m i l a r rate i n prawns from a l l treatments up to day 6 (Figure 23). A f t e r the f i r s t 6 days of storage, hypoxanthine concentration increased very r a p i d l y i n the NMAP prawns u n t i l day 9 a f t e r which increases i n hypoxanthine concentration occurred slowly. rate  A f t e r day 6, hypoxanthine concentration increased a t a slower  i n the CMAP prawns than  i n the NMAP prawns.  These changes i n  hypoxanthine concentration i n each treatment were s i g n i f i c a n t during 6 days storage as w e l l as 18 days storage (Table 6 ) . there  were  significant  differences  (P<0.01)  On day 12 and day 18,  among  the hypoxanthine  concentrations i n the CMAP and the NMAP prawns (Table 7 ) . 4.1.6.  Trimethylamine-nitrogen concentration  TMAN concentration increased very slowly i n the prawns from each treatment up to day 6 (Figure 24). tion  then  increased  rapidly  until  I n the NMAP prawns, TMAN concentrathe l a s t  day o f storage.  TMAN  concentration i n the CMAP prawns slowly increased up to day 9 and then increased r a p i d l y at a rate s i m i l a r to that observed f o r the NMAP prawns u n t i l the l a s t day of storage.  The NMAP prawns had higher concentrations  of TMAN than the CMAP prawns at the end of T r i a l 1. Changes i n the TMAN concentration were not s i g n i f i c a n t i n the CMAP prawns but s i g n i f i c a n t i n the  c o n t r o l and the NMAP prawns during  the f i r s t  6 days o f storage  (P<0.01, Table 6 ) . During 18 days storage, TMAN concentration i n the CMAP as w e l l as the NMAP prawns changed s i g n i f i c a n t l y  (P<0.01, Table 7 ) .  S i g n i f i c a n t d i f f e r e n c e s (P<0.01) among the TMAN concentrations of these (CMAP and NMAP) treatments  were  found  (P<0.01, Table  6) from day 9  81  F  S  r  2  la  n  0nC  ntrati<,nS  f  St  a ir o\ L contro\ :„dnitrogen : ° ^ °™* , caroon^«bo^S" d i o x i d e , and atmospheres at 1°C  82  Figure 24. T r i a l 1: Trimethylamine-nitrogen concentrations of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  83  onwards. TMAN concentrations of the NMAP prawns were s i g n i f i c a n t l y higher (P<0.05) than those of the c o n t r o l and the CMAP prawns. 4.1.7.  Sensory e v a l u a t i o n  Changes i n the sensory c h a r a c t e r i s t i c s evenly developed.  among treatments were not  Samples having an o v e r a l l sensory score o f less then  5.00 were considered to be of an unacceptable q u a l i t y .  A t the end o f the  storage time, the o v e r a l l sensory scores o f above 5.00 i n d i c a t e d that a l l prawns except c o n t r o l samples were s t i l l acceptable from a sensory point of  view (Figure 25).  However, as shown i n Figure 26, the NMAP prawns  could have been rejected on day 9 of storage based on the scores of raw prawn meat odour which i s one of the prime freshness c r i t e r i a often used by customers.  Furthermore, a l l samples might have also been rejected  based on the scores of cooked meat odour as shown i n Figure 27.  There  was no c l e a r trend of e i t h e r the NMAP or the CMAP being more capable of maintaining prawn sensory c h a r a c t e r i s t i c s except f o r colour and texture of  cooked meat, where the NMAP prawns generally had b e t t e r scores than  the CMAP prawns (Figures 28 and 29).  Scores f o r raw prawn meat colour  and scores f o r cooked prawn meat flavour were as shown i n Figures 30 and 31 r e s p e c t i v e l y . The r e s u l t s from the Friedman two-way ANOVA on these sensory data (Table 8) showed that the panel d i d not detect any s i g n i f i c a n t changes i n prawns from each treatment with storage l i f e up to day 6 except f o r the cooked prawn meat colour of the NMAP prawns.  The panel also d i d not  detect any s i g n i f i c a n t changes i n sensory c h a r a c t e r i s t i c s i n prawns from each treatment with storage l i f e up to day 18 except f o r the raw prawn meat odour and the cooked prawn meat odour of the NMAP prawns.  The  84  B Control  — • — CMAP  O O  A  if)  NMAP  o w c  Q) Ul  >  o  12  15  18  Days of Storage  Figure 25. T r i a l 1: O v e r a l l sensory scores of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  85  B Control  (D i_  — • —  o o  00  CMAP  i_  D O  — ± —  O  NMAP  "O  •4->  O  C CL o cn  12  15  18  Days of Storage  Figure 26. T r i a l 1: Scores f o r raw prawn meat odour of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres a t 1°C  86  Figure 27. T r i a l 1: Scores f o r cooked prawn meat odour of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  87  B  Control  i_  O O  — • —  00  CM/AP  D  o o o  — • — NMAP  a (D  2  CL  T> (D  o o o  9  12  15  18  Days of Storage  Figure 28. T r i a l 1: Scores f o r cooked prawn meat colour of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  88  Figure 29. T r i a l 1: Scores f o r cooked prawn meat texture of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at  X c  89  Figure 30 T r i a l 1: Scores f o r raw prawn meat colour of prawns stored under aerobrc c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  90  at  91  Table 8. F - s t a t i s t i c s ( a ) from Tukey's t e s t on data o f T r i a l 2 (data o f 12 day)  All Indicators (b)  Control  Exudate  CMAP  NMAP  NV  NV  NV (c)  Treatments NS (d)  PH  39..712 **  24..516 **  15..838 **  19..615 **  TSA  88..706 **  15..673 **  56..071 **  3..570 *  TSN  87..449 **  16..708 **  49,.750 **  NS  SPA  301..252 **  72..660 **  199..290 **  NS  SPN  318..807  **  NS  TMAN  187..932 **  5..557 *  31..613 **  NS  TVBN  17..966 **  12..693 **  16..430 **  3..625 *  WSP  1065..782 **  9..946 **  **  4..136 *  SSP  14..386 **  11..547 **  8..324 **  8..188 **  4..659 *  8,.179 **  12..728 **  4..867 *  16..413 **  14..274 **  L  **  26..914  **  218..110  31..773  a  NS  NS  b  NS  NS  NS  NS  RC  NV  NV  NV  NS  RO  NV  NV  NV  NS  CC  NV  NV  NV  CO  NV  NV  NV  NS  CF  NV  NV  NV  NS  CT  NV  NV  NV  NS  Overall  NV  NV  NV  NS  (a)  * = s i g n i f i c a n t a t 5% l e v e l ** = s i g n i f i c a n t a t 1% l e v e l  (b)  See abbreviation l i s t  (c)  One or more of groups has no variance  (d)  No s i g n i f i c a n c e  10..414 **  92  panel, however, d i d detect differences i n the cooked prawn meat colour, the raw prawn meat odour, and the raw prawn meat colour among prawns of d i f f e r e n t treatments during the f i r s t 12 days o f storage. In the two-way a n a l y s i s of variance on each sensory significant  d i f f e r e n c e s among judges  characteristic  were  evident  characteristic,  i n every  sensory  (P<0.01).  4.1.8. Hunter L, a, b values While the Hunter L value of the c o n t r o l prawns remained the same during the f i r s t 6 day of storage, i t increased i n both the CMAP and the NMAP prawns i n a s i m i l a r manner with the CMAP having a s l i g h t l y higher value by the end of 18 days of storage (Figure 32).  I n each  treatment,  the Hunter L value changed s i g n i f i c a n t l y (P<0.01) i n the CMAP and the NMAP but not the c o n t r o l prawns during 6 days storage (Table 6 ) .  During  18 days storage, the Hunter L value changed s i g n i f i c a n t l y (P<0.01) i n both the CMAP and the NMAP prawns (Table 6 ) . storage,  the Hunter  significantly different  L  values  among  On day 3 and day 6 of the  a l l three  (P<0.05, Table 7 ) .  treatments  were  A f t e r that,, there were no  s i g n i f i c a n t differences i n the Hunter L value (between the CMAP and the NMAP prawns). While the Hunter a value of the c o n t r o l prawns changed very it  little,  increased i n both the CMAP and the NMAP prawns (Figure 33) .  The  Hunter a value of the CMAP prawns increased u n t i l day 6 and then  just  s l i g h t l y increased u n t i l the end of storage.  The Hunter a value of the  NMAP prawns d r a m a t i c a l l y increased u n t i l day 9 and then remained about the same u n t i l the end of storage.  During 6 days storage, changes i n the  Hunter a value were s i g n i f i c a n t i n the CMAP prawns (P<0.01, Table 6 ) . At  42  37' 0  3  6  9  12  15  18  Days of Storage  Figure 32. T r i a l 1: Hunter L values of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  94  0  3  6  9  12  15  18  Doys of Storage  Figure 33. T r i a l 1: Hunter a values of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  95  12.5  10.0'  :  0  3  6  9  12  15  18  Days of Storage  Figure 34. T r i a l 1: Hunter b values of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  96  the same storage p e r i o d , however, changes i n the Hunter a value i n the NMAP prawns were marginally s i g n i f i c a n t  (P=0.058).  The Hunter a values  of the CMAP and the NMAP prawns changed s i g n i f i c a n t l y (P<0.01) during 18 days  storage. When comparing  significant  the Hunter  a values  among  treatments,  differences were found on day 6, day 12, and day 15 (Table  7). The Hunter b value increased i n a l l prawn samples evaluated (Figure 34).  The NMAP prawns had s l i g h t l y higher Hunter b values than the CMAP  prawns during most o f the storage t r i a l .  The Hunter b value i n the NMAP  prawns increased u n t i l day 9 and then slowly decreased u n t i l the end of storage.  During 6 days storage of each treatment, the Hunter b value of  prawns changed s i g n i f i c a n t l y  (Table  6).  Significant  changes of the  Hunter b values i n the CMAP and the NMAP prawns were also observed during 18 days storage.  But there was no s i g n i f i c a n t d i f f e r e n c e i n the Hunter b  value among treatments on any sampling day throughout the storage. 4.2.  Trial 2 In T r i a l 2 of t h i s study, sampling was c a r r i e d out every 4 days up  to  day 28 f o r a l l treatments  (the c o n t r o l ,  the CMAP, and the NMAP  prawns). 4.2.1.  Headspace gas composition  Changes i n the headspace gas composition i n the CMAP and the NMAP bags are i l l u s t r a t e d i n Figures 35 and 36 r e s p e c t i v e l y .  A slight in-  crease i n carbon dioxide concentration gradually took place i n the NMAP bags,  similar  to that  observed  i n Trial  1.  Changes  i n nitrogen  concentration i n the bags were p r o p o r t i o n a l to the changes i n the carbon  97  DayO  Day8  Day16  Day28  Figure 35. T r i a l 2: Concentrations of carbon d i o x i d e , oxygen, and nitrogen gas i n the CMAP bags stored at 1°C  98  100  93.21  91.04  86.54  80  C o CL  60  40  20 8.86 0  0.9^6 DayO  12.95 6.67  MM Day8  i.5'  Day16  Day28  Figure 36. T r i a l 2: Concentrations of carbon d i o x i d e , oxygen, and nitrogen gas i n the NMAP bags stored at 1°C  99  dioxide concentration.  Carbon dioxide concentration i n the CMAP bags  dropped between day 0 and 4 and l a t e r slowly increased. Carbon dioxide concentrations i n the CMAP bags during storage fered s i g n i f i c a n t l y  (F=17.472, P<0.01) with  highest and s i g n i f i c a n t l y d i f f e r e n t  dif-  that of day 0 being the  from the r e s t .  A significant  dif-  ference i n carbon dioxide concentration i n the NMAP bags during storage was also found (F=8.922, P<0.01). 4.2.2.  Tissue pH  Changes i n the tissue pH of prawns are shown i n Figure 37. The prawns, at the time of packing, had a pH of about 7.5.  After a slight  i n i t i a l drop, the pH of the c o n t r o l prawns increased over storage  time.  The pH of the NMAP prawns remained i n the range o f 7.3 to 7.5 over the 28 days storage p e r i o d . of storage.  pH of the CMAP prawns dropped to 6.5 w i t h i n 4 days  The CMAP prawns increased s l i g h t l y to pH 6.7 by day 12 where  they remained f o r the duration of the storage storage, there was a s i g n i f i c a n t  trail.  During  12 days  change (P<0.01) i n prawn t i s s u e pH i n  each treatment (Table 9 ) . Differences i n t i s s u e pH among treatments were significant  on every sampling day during 12 days storage (P<0.01, Table  9). Tissue pH values of the CMAP prawns were always s i g n i f i c a n t l y (P<0.05)  than  those  of the NMAP  prawns  which  were  also  lower always  s i g n i f i c a n t l y lower (P<0.05) than those of the c o n t r o l prawns. 4.2.3. Exudate formation Changes i n exudate formation i n prawns are shown i n Figure 38. and f a l l o f exudate values was found i n t h i s t r i a l .  Rise  This was probably  the r e s u l t of the preparation procedure used to remove the roe from the  100  Figure 37. T r i a l 2: Inner t i s s u e pHs of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  Table 9.  F - s t a t i s t i c s (a) from ANOVA on data 12 days storage of T r i a l 2  Variables (b)  Exudate (c) pH TSA TSN SPA SPN TMAN TVBN WSP SSP L a b  Variables  Exudate pH TSA TSN SPA SPN TMAN TVBN WSP SSP L a b  (a) (b) (c)  Control  3..847 39..712 88..706 88..449 301..252 318..807 184..932 17..966 1065..782 14..386 4..659 1. .083 3..059  CMAP  * ** ** ** ** ** ** ** ** ** **  0.318 24. 516 15. 673 16. 708 72. 660 26. 914 5.557 12. 693 9. 946 11.547 8.179 4.040 3.504  Day4  Day8  0..750 283 .978 31..903 32 .411 38 .661 12..226 2 .448 4..732 15,.322 8,.439 0..300 4,.579 0..204  12..761 146..184 26..760 19..598 712..262 37..081 10..265 5..463 50..944 8..037 11..838 10..633 6..588  ** ** ** ** ** ** *  NMAP  ** ** ** * ** * ** ** ** **  2 .507 15 .838 56 .071 49 .750 199 .290 218 .110 31 .613 16 .430 31 .773 8 .324 12 .728 16 .413 0 .316  ** ** ** ** ** ** ** ** ** ** **  Dayl2  ** ** ** ** ** ** * * ** * ** * *  0..727 48..533 95..573 158..447 115..076 60..046 10..822 15..655 174..210 38..249 41..885 48..473 2..676  * = s i g n i f i c a n t at 5% l e v e l ** = s i g n i f i c a n t at 1% l e v e l See Abbreviation L i s t Only data of day 4 to day 12; no exudate on day 0  ** ** ** ** ** * ** ** ** .JUJL. **  102  16  Days of Storage  Figure 38. T r i a l 2: Exudate volumes of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  103  prawns.  Roe were removed by opening up the carapaces at the abdominal  area, and scraping them o f f with a s p a t u l a .  This procedure, although  performed c a r e f u l l y ,  damaged the carapaces and exposed the abdominal  t i s s u e of the prawn.  Generally, more exudate was observed i n the CMAP  prawns than i n the NMAP or c o n t r o l prawns. A s i g n i f i c a n t change (P<0.05) i n exudate volume was found i n the c o n t r o l prawns during 12 days storage (Table 9 ) .  Only on day 8, differences i n exudate volume among these  treaments were s i g n i f i c a n t (P<0.01, Table 9 ) . 4.2.4. Microbiology Figures 39 to 40 show the changes i n TSA and TSN the CMAP, and the NMAP prawns r e s p e c t i v e l y .  o f the c o n t r o l ,  The highest TSA and TSN were  found i n the c o n t r o l prawns followed by the NMAP and the CMAP prawns respectively.  On the whole, aerobic psychrotrophic b a c t e r i a grew more  slowly i n the NMAP and CMAP prawns with slowest growth observed i n the CMAP prawns. Changes i n SPA and SPN of the c o n t r o l , the CMAP, and the NMAP prawns were shown i n Figures 41 to 42 r e s p e c t i v e l y . (day  0) had a s i g n i f i c a n t l y  higher  The raw m a t e r i a l prawns  population  of sulphide-producing  aerobes than sulphide-producing anaerobes (T=4.770, P=0.041).  By the end  of 28 days storage, the aerobic and anaerobic populations of sulphideproducing  psychrotrophic  bacteria  were  similar  i n a l l treatments.  Aerobic sulphide-producing psychrotrophic b a c t e r i a grew at a s i m i l a r rate i n the c o n t r o l and the NMAP prawns, with the population l e v e l l i n g o f f to about 10  5  cfu/gm by day 8.  In the CMAP prawns, aerobic  sulphide-  producing psychrotrophic b a c t e r i a grew at a slower rate and l e v e l l e d o f f slowly by the end of storage. Anaerobic sulphide-producing psychrotrophic  104  Figure 39. T r i a l 2: T o t a l aerobic psychrotrophic b a c t e r i a l counts of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and n i t r o g e n atmospheres at 1°C  105  Figure 40. T r i a l 2: T o t a l anaerobic psychrotrophic b a c t e r i a l counts of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and n i t r o g e n atmospheres at 1°C  106  Figure 41. T r i a l 2: Total aerobic sulphide-producing psychrotrophic b a c t e r i a l counts of prawns stored under aerobic c o n t r o l , carbon dioxide, and nitrogen atmospheres at 1°C  107  Figure 42. T r i a l 2: Total anaerobic sulphide-producing psychrotrophic b a c t e r i a l counts of prawns stored under aerobic c o n t r o l , carbon dioxide, and nitrogen atmospheres at 1°C  108  b a c t e r i a grew more r a p i d l y than aerobic sulphide-producing psychrotrophic b a c t e r i a during the f i r s t 12 days of storage i n the c o n t r o l and the NMAP prawns.  Anaerobic sulphide-producing psychrotrophic b a c t e r i a grew much  l e s s r a p i d l y i n the CMAP prawns, but the population by day 28 was s i m i l a r to that observed f o r the NMAP prawns. Changes i n TSA, TSN, SPA, and SPN during 12 day storage of each treatment were s i g n i f i c a n t (P<0.01, Table 9 ) . S i g n i f i c a n t d i f f e r e n c e s i n TSA, as w e l l as TSN, SPA, and SPN among treatments were also observed on each sampling day during 12 days storage (P<0.01, TAble 9 ) . 4.2.5.  Trimethylamine-nitrogen concentration  Changes i n TMAN concentrations  are shown i n Figure 43. The TMAN  concentration increased very slowly i n the c o n t r o l and the NMAP prawns up to day 4, and i n the CMAP prawns up to day 8. TMAN concentration i n the NMAP prawns then r a p i d l y increased u n t i l the l a s t day of storage.  After  day 8, the TMAN concentration i n the CMAP prawns r a p i d l y increased u n t i l the end of storage.  The TMAN concentrations i n the c o n t r o l prawns up to  day 16 were lower than those i n the NMAP prawns but higher than those i n the CMAP prawns.  A f t e r day 16, the TMAN concentration i n the c o n t r o l  prawns suddenly decreased before s t a r t i n g to increase again u n t i l the end of storage.  At the end of storage, the highest concentration of TMAN was  found i n the NMAP prawns followed by the CMAP and the c o n t r o l prawns respectively. Changes i n TMAN concentration i n these prawns during 12 storage were s i g n i f i c a n t i n each treatment (Table 9 ) . While no s i g n i f i c a n t d i f f e r e n c e i n TMAN concentration among treatments was observed on day 0, s i g n i f i c a n t differences were found on day 8 and day 12 (Table 9).  109  90  0  4  8  12  16  20  24  28  Days of Storage  Figure 43. T r i a l 2: Trimethylamine-nitrogen concentrations of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and n i t r o g e n atmospheres at 1°C  110  4.2.6.  Total v o l a t i l e basic nitrogen concentration  The TVBN concentration i n a l l prawns (Figure 44) increased s l i g h t l y at a s i m i l a r rate up to day 4.  A f t e r day 4, i t increased r a p i d l y a t a  s i m i l a r rate i n the c o n t r o l and the NMAP prawns.  A f t e r day 20, the TVBN  concentration i n the NMAP prawns dropped d r a m a t i c a l l y while  the TVBN  concentration i n the c o n t r o l prawns continued to increase, but a t a much slower  rate than before.  increased  gradually  until  The TVBN concentration i n the CMAP prawns day 16, and then  remained a t about 100  mgN/lOOgm t i s s u e u n t i l the end of storage. Changes  i n TVBN concentration i n each treatment during  12 days  storage were s i g n i f i c a n t (P<0.01, Table 9 ) . But s i g n i f i c a n t d i f f e r e n c e s i n TVBN concentration among treatments were observed only on day 8 and day  12  (Table  significantly  9).  TVBN  concentrations  (P<0.05) lower  than  o f the CMAP prawns  were  those o f the NMAP and the c o n t r o l  prawns during 12 days storage. 4.2.7.  Soluble p r o t e i n s  Changes i n the concentrations of WSP and SSP o f the c o n t r o l , the CMAP, and the NMAP prawns  are shown  i n Figures  45 to 46.  The  concentration of SSP was higher than that of WSP a t the beginning of storage.  Generally, SSP concentration increased a t f i r s t , then gradually  decreased with the storage time i n a l l prawns while the WSP concentration increased gradually and then storage.  decreased continuously u n t i l the end of  The highest concentration of the WSP was i n the c o n t r o l prawns  followed by the NMAP and the CMAP prawns r e s p e c t i v e l y .  I n contrast to  the development of the WSP, the highest concentration o f the SSP was i n the CMAP prawns followed by the NMAP and the c o n t r o l prawns r e s p e c t i v e l y .  Ill  Figure 44. T r i a l 2: Total v o l a t i l e basic nitrogen concentrations of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  112  Figure 45. T r i a l 2: Water-soluble p r o t e i n concentrations of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and n i t r o g e n atmospheres at 1°C  113  Figure 46. T r i a l 2: S a l t - s o l u b l e p r o t e i n concentrations of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  114  During the l a s t week of storage of the c o n t r o l prawns, however, there was a  sudden  increase  i n the SSP concentration  which,  after  day 24,  immediately decreased u n t i l the end of storage. Changes  i n WSP and SSP f o r prawns i n each treatment group were  s i g n i f i c a n t during 12 days storage (P<0.01, TAble 9 ) . WSP as w e l l as SSP of these  treatments  were s i g n i f i c a n t l y d i f f e r e n t on each sampling day  during 12 days storage (Table 9 ) . 4.2.8.  Sensory evaluation  By using an o v e r a l l sensory score of more than or equal to 5.00 as an i n d i c a t i o n of the acceptable  q u a l i t y of the prawns, the scores i n  Figure 47 i n d i c a t e d that the panel r e j e c t e d the c o n t r o l prawns on day 8 and the CMAP and the NMAP prawns on day 12.  Scores f o r each sensory  c h a r a c t e r i s t i c (RC, RO, CC, CO, CF, and CT) were as shown i n Figures 48 to  53 r e s p e c t i v e l y .  For the CMAP prawns at day 12, however, the average  scores f o r the raw prawn meat odour and colour and the cooked prawn meat colour were s t i l l i n the acceptable range.  On day 8, while the average  score f o r raw prawn meat odour of the CMAP prawns was s t i l l  acceptable,  the scores f o r the c o n t r o l and the NMAP prawns were i n the r e j e c t i o n range (Figure 48).  Raw prawn meat colour of the NMAP prawns was s t i l l  acceptable by day 12 while raw prawn meat colour of the c o n t r o l and the CMAP prawns were considered unacceptable  (Figure 4 9 ) .  Cooked prawn meat  odour of a l l samples were r e j e c t e d by day 4 (Figure 50). On day 8, while the cooked prawn meat colour of the NMAP and the c o n t r o l were s t i l l quite acceptable, the cooked prawn meat colour of the CMAP prawns were r e j e c t e d (Figure 51). On day 8, the cooked prawn meat flavour of the CMAP and the NMAP prawns were s t i l l acceptable but that of the c o n t r o l prawns was not  115  Figure 47. T r i a l 2: O v e r a l l sensory scores of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  116  Figure 48. T r i a l 2: Scores f o r raw prawn meat colour of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  117  Figure 49. T r i a l 2: Scores f o r raw prawn meat odour of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  118  Figure 50. T r i a l 2: Scores f o r cooked prawn meat colour of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  119  Figure 51. T r i a l 2: Scores f o r cooked prawn meat odour of prawns stored under aerobic c o n t r o l , carbon dioxide, and nitrogen atmospheres at 1°C  120  Figure 52. T r i a l 2: Scores f o r cooked prawn meat f l a v o u r of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and n i t r o g e n atmospheres at 1°C  121  <D i_ O O  8  Ul fi?  7  D X  6  O  5 C  4  CL "O  0)  _x" o o  o  3 2  0  8  12  Days of Storage  F  J£?« ° P « ~ meat texture of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at T r i a l  2 :  u  S c  r e S  f o r  c o o k e d  Table 10. F - s t a t i s t i c s (a) from Friedman two-way ANOVA on sensory data of T r i a l 2 I.  Data o f 12 days storage  Variables (b) RC RO CC CO CF CT II.  6.600 12.250 ** 10.350 ** 10.100 * 11.450 ** 1.020  (a) (b)  CMAP  NMAP  10.350 * 3.350 4.500 3.240 4.020 5.940  4.500 8.150 * 2.600 6.150 6.180 3.000  Data of i n d i v i d u a l sampling day  Variables RC RO CC CO CF CT  Control  Day4  Day8  Dayl 2  1.750 1.583 4.083 0.083 0.583 0.083  1.583 2.083 5.583 1.900 0.900 5.375  1.900 7.000 * 2.083 1.083 6.583 * 1.583  * = s i g n i f i c a n t a t 5% l e v e l ** = s i g n i f i c a n t at 1% l e v e l See Abbreviation L i s t  123  (Figure 52).  By day 8, cooked prawn meat texture score f o r the CMAP  prawns f e l l into the r e j e c t i o n range but those o f the c o n t r o l and the NMAP prawns were s t i l l i n the acceptable range (Figure 53). The r e s u l t s from Friedman two-way ANOVA on these sensory data (Table 10) showed that the panel detected s i g n i f i c a n t changes i n : raw prawn meat odour, cooked prawn meat c o l o u r , odour, and flavour  o f the c o n t r o l  prawns; raw prawn meat colour of the CMAP prawns; and raw prawn meat odour of the NMAP prawns.  The panel detected only the s i g n i f i c a n t  (P<0.05) differences i n raw prawn meat odour and i n cooked prawn meat flavour of prawns among treatments on day 12 of storage. analysis  of variance  differences among judges  on  each  sensory  I n the two-way  characteristic,  were evident i n every  sensory  significant characteristic  (P<0.01). 4.2.9.  Hunter L, a, b values  The Hunter L values (Figure 54) of a l l prawns were s i m i l a r up to day 4.  Then the Hunter L value of the c o n t r o l prawns s t a r t e d to decrease  gradually, consistently  while  those  and those  o f the CMAP  prawns  o f the NMAP prawns  continued  to increase  increased only  slightly.  Changes i n the Hunter L value of each treatment were s i g n i f i c a n t (P<0.01, Table 9 ) .  But s i g n i f i c a n t differences (P<0.01) i n the Hunter L value  among treatments were observed on day 8 and day 12. The Hunter a value (Figure 55) was highest i n the NMAP prawns f o l lowed by the CMAP and the c o n t r o l prawns r e s p e c t i v e l y .  The Hunter a  values of the NMAP and the CMAP prawns increased but the Hunter a values of the c o n t r o l prawns decreased during storage.  During 12 days storage,  changes i n the Hunter a. value were s i g n i f i c a n t only i n the NMAP prawns  124  Figure 54. T r i a l 2: Hunter L values of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  125  Figure 55. T r i a l 2: Hunter a values of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  126  Figure 56. T r i a l 2: Hunter b values of prawns stored under aerobic c o n t r o l , carbon d i o x i d e , and nitrogen atmospheres at 1°C  127  (P<0.01, Table 9 ) .  S i g n i f i c a n t differences i n the Hunter a value among  treatments were found on day 8 and day 12. The Hunter b values  (Figure 56) of the c o n t r o l and the CMAP prawns  changed s l i g h t l y during the f i r s t 4 days of storage until  day 8.  dropped  A f t e r t h a t , the Hunter b values  dramatically  increase.  while  those  o f the CMAP  and then  increased  of the c o n t r o l prawns prawns  continued  The Hunter b values o f the NMAP prawns increased  to  gradually  u n t i l day 4 and decreased gradually a f t e r day 4 back to the i n i t i a l value by day 12.  No s i g n i f i c a n t changes (P>0.05) i n the Hunter b value were  found among treatments during storage.  A s i g n i f i c a n t d i f f e r e n c e i n the  Hunter b value among treatments was observed on day 8 only. 4.2.10.  Factor analysis  In the r e s u l t s from f a c t o r a n a l y s i s , v a r i a t i o n i n the m u l t i v a r i a t e data was summarized by 4 f a c t o r s or p r i n c i p a l components.  The f i r s t  extracted f a c t o r accounted f o r 59.07% o f the data v a r i a n c e .  The f i r s t  two  extracted f a c t o r s accounted f o r 80.08% o f the data variance.  The  f i r s t three extracted f a c t o r s accounted f o r 90.90% o f the data variance. All  four extracted f a c t o r s accounted f o r 100.00% o f the data variance.  The  sorted rotated f a c t o r loadings  broad f a c t o r , contained colour flavour  score score  (Table  11) shows that Factor  1, a  a l l m i c r o b i o l o g i c a l v a r i a b l e s , odour score and  of raw prawn meat, TMAN concentration, o f cooked prawn meat, WSP, and o v e r a l l  Factor 2 described colour: Hunter L and a values and SSP.  odour score and sensory  score.  Factor 3 was a  measure of colour and texture of cooked prawn meat, exudate volume, and pH.  Factor 4 was a measure of Hunter b value and TVBN concentration.  Table 11. Sorted rotated factor loadings from factor (a) analysis of data  Variables TSA TSN RO SPA SPN TMAN CF WSP OVERALL CO RC a SSP L CC DRIP pH CT b TVBN  (b)  FACT0R1  FACT0R2  1.000 0.980 -0. 950 0.946 0.908 0.862 -0. 857 0.833 -0. 830 -0. 823 -0. 765 0.000 0.000 0.000 0.000 0.000 0.651 -0. 311 0.000 0.505  0..000 0..000 0..000 0..000 0..000 0..290 0..000 -0..397 0..000 0..000 0..000 0 .933 0..881 0..858 0 .327 0,.000 -0..378 0,.000 0..000 0..000  FACT0R3 0 .000 0 .000 0 .000 0 .000 0 .000 0 .000 0 .270 0 .000 0 .342 0 .306 0 .000 0 .000 0 .000 0 .000 0 .825 -0 .822 0 .691 0 .626 0 .000 0 .000  FACT0R4 0..000 0.,000 0..000 0..000 0..000 0..000 0.,000 0..000 0..000 0..000 -0..581 0..000 0..000 0..325 -0..272 0..000 0..000 0..319 0..801 -0..634  The above factor loading matrix has been rearranged so that the columns appear i n decreasing order of variance explained by factors. The rows have been rearranged so that for each successive f a c t o r , loadings greater than 0.5000 appear f i r s t . Loadings less than 0.2500 have been replaced by zero. See Abbreviation L i s t  Table 12. Rotated f a c t o r loadings from f a c t o r analysis of data Variables EXUDATE PH TSA TSN SPA SPN TMAN TVBN WSP SSP L a b OVERALL RC RO CC CO CF CT  (a)  FACT0R1  FACT0R2  FACT0R3  FACT0R4  0..099 0..651 1..000 0..980 0..946 0..908 0..862 0..505 0..833 -0. .022 0..048 0..096 0..190 - 0 . .830 - 0 . .765 - 0 . .950 - 0 . .197 - 0 . ,823 - 0 . .857 - 0 . .311  0..186 -0. .378 0..050 0,.066 0..109 0..158 0..290 -0. .013 -0. .397 0..881 0..858 0..933 0..137 0..099 0..033 0..051 0..327 0..000 0..179 -0. .196  -0 .822 0 .691 0..085 0 .019 -0 .043 -0. .121 0 .138 -0. .128 -0. .024 -0, .090 - o . .105 0..073 -0. .174 0..342 -0. .091 0..006 0..825 0..306 0..270 0..626  -0. .012 - 0 . .035 0..045 0..071 0..102 - 0 . .065 -0. .210 - 0 . .634 - 0 . .108 - 0 . .157 0..325 0..025 0..801 - 0 . .114 - 0 . .581 0..022 - 0 . .272 0..019 - 0 . .042 0..319  See Abbreviation L i s t  130  1.0  Hunter a S S  0.5  Hunter L  H CC TMAN  CM  °  Exudate Hunter b  CF  o.o -J  RO  Overall  SPN TVBN  O  a  CT  Li_ pH  w  s  p  -0.5  1.0  1  •1.0  '  I  I  I  -0.5  I  I  I  1  |  I  1  0.0  1  1  1 0.5  I  1  1  1  1 1.0  Factor 1  Figure 57. T r i a l 2: P l o t s of Factor 1 against Factor 2 of r o t a t e d factor loadings from the f a c t o r analysis on data of 12 days storage of Trial 2  131  The  rotated  f a c t o r loadings  shown i n Table 12.  from factor a n a l y s i s o f the data was  The p l o t of the rotated f a c t o r loadings f o r Factor 1  versus Factor 2 i s shown i n Figure 57 and indicates c l u s t e r i n g o f several Groups o f v a r i a b l e s : odour of raw and cooked prawn meat, colour of raw and  flavour  concentration,  of cooked prawn meat, and o v e r a l l and Hunter L and a values;  value; WSP concentration  sensory  scores;  SSP  exudate volume and Hunter b  and trimethylamine-nitrogen concentration; and  a l l m i c r o b i o l o g i c a l data. 4.2.11.  Stepwise discriminant analysis  Stepwise discriminant analysis on the " m u l t i v a r i a t e data" with TMAN concentration  as the grouping v a r i a b l e r e s u l t e d i n 9 v a r i a b l e s .  These  v a r i a b l e s were SPA, pH, TVBN, CO, SSP, CF, WSP, CT, and Hunter L value ranked i n descending order of U - s t a t i s t i c .  The smaller the U - s t a t i s t i c  becomes, the c l o s e r i s the c l a s s i f i c a t i o n coming to p e r f e c t i o n (Powers and  Ware, 1986).  obtained from t h i s  Classification  matrix and Jackknife  stepwise discriminant  classification  analysis were 100% and 94.4%  correct. When considering  the p r a c t i c a l i t y , using 9 t e s t s to evaluate prawn  q u a l i t y i s too time-consuming, laborious, and c o s t l y .  To overcome t h i s  s i t u a t i o n , a minimum number o f t e s t must be s e l e c t e d .  I n a case where  one t e s t i s h i g h l y c o r r e l a t e d with another or moderately c o r r e l a t e d with several and these other t e s t s have already been included i n the process, the c o r r e l a t e d terms would not be providing new information  useful f o r  d i s c r i m i n a t i o n but only information p a r t i a l l y redundant with that already a v a i l a b l e (Powers and Ware, 1986).  They commented that a v a r i a b l e which  by i t s e l f was not a great discriminator but which was not w e l l c o r r e l a t e d  132  with  any other v a r i a b l e already i n use was more l i k e l y to add to the  d i s c r i m i n a t i o n power than one which was c o r r e l a t e d . stepwise discriminant variables.  Therefore, more  analyses were c a r r i e d out on many combinations o f  The combination of SPA and pH produced the highest (97.2%)  success i n both c l a s s i f i c a t i o n matrix and Jackknife f a c t , the r e s u l t from the Jackknife  classification.  In  c l a s s i f i c a t i o n was b e t t e r than when  a l l v a r i a b l e s ( t e s t s ) were used i n the a n a l y s i s . As a r e s u l t o f the stepwise discriminant analysis on pH and SPA data using the TMAN concentration as the grouping v a r i a b l e , the c o e f f i c i e n t s for  2 canonical  variables  were produced  as shown  i n the f o l l o w i n g  equations: Canonical v a r i a b l e 1 = 3.00114 [pH] - 1.99857 [SPA] - 14.06103 Canonical v a r i a b l e 2 = 2.45639 [pH] - 0.06084 [SPA] - 18.10119 The c o e f f i c i e n t s of v a r i a b l e s describe the two canonical  variables  that are used f o r v i s u a l d i s c r i m i n a t i o n of the group i n two-dimensional space.  The p l o t was as i l l u s t r a t e d i n Figure 59. The r e s u l t produced  only 1 i n c o r r e c t c l a s s i f i c a t i o n (out of 36 cases) which was i n d i c a t e d by the c i r c l e on the p l o t .  This p l o t belonged to the data o f the second bag  of the CMAP prawns of day 8.  133  3.0  CM 2.0 -  cc c c c  CD <  < >  i-o  cc BP  o.o  _l <  O  i.o —  < -2.0  -3.0  1 I I I I i—r~i—i—r~i—r~ 6.0  -4.0  -2.0  0.0  I I I I I I I I I 2.0  4.0  6.0  CANONICAL VARIABLE 1  Figure 58. T r a i l 2: Canonical p l o t s of prawn samples c l a s s i f i e d prawns by stepwise discriminant analysis of pH and SPA A = Grade I: < 1.00 mg TMAN/ 100 gm t i s s u e B = Grade I I : 1.01 to 5.00 mg TMAN/ 100 gm t i s s u e C = Grade I I I : > 5.00 mg TMAN/ 100 gm tissue  134  5. DISCUSSION  135  5.  DISCUSSION  5.1.  Headspace gas compositions i n the bags  The  results from the gas chromatographic analysis showed an immedi-  ate decrease i n the concentration of carbon dioxide  i n the CMAP bags.  This reduction of the carbon dioxide concentration i n the CMAP bags was mainly the r e s u l t of a portion of carbon dioxide gas d i s s o l v i n g into the liquid  phase of prawn tissue.  dioxide  i s more  temperature  soluble  decreases.  Compared to most other  i n water.  Gases  Its solubility  transmission  through  gases, carbon increases  the f i l m  as  would be  n e g l i g i b l y small since the f i l m i s p r a c t i c a l l y gas-impermeable.  During  storage of the prawns, the carbon dioxide concentration i n the headspace atmosphere  increased  reactions.  Collapse or deformation of the CMAP bags occurred  study  as a result  due  to microbial  of carbon  dioxide  respiration  or  biochemical i n this  d i s s o l v i n g i n the prawn muscle  tissue. On the contrary, there was no such dramatic changes i n the headspace gas composition i n the NMAP bags.  The concentration of carbon dioxide i n  the NMAP bags s l i g h t l y increased as a result of microbial a c t i v i t i e s and tissue enzymatic reactions. in  The changes of the nitrogen  the NMAP bags were mainly  proportional  concentrations  to the changes  of carbon  dioxide levels i n the bags.  5.2.  T i s s u e pHs  The  i n i t i a l pH of the pink prawns i n this study was i n agreement  with the findings of F l i c k and Lovell (1972) and Bailey et al. , (1956).  136  The  immediate drop i n the tissue pH of the CMAP samples happened at a  time c o i n c i d i n g with the reduction of carbon dioxide concentrations i n the  CMAP bags.  This  phenomenon was also  Lannelongue et al., (1982).  reported  i n a study by  An absence o f the i n i t i a l  pH drop was  evident i n the c o n t r o l prawns of T r i a l 1 but not i n T r i a l 2. An i n i t i a l pH drop i n the NMAP prawns occurred i n both t r i a l s i n t h i s study.  This  may be a r e s u l t o f g l y c o l y s i s or the carbon dioxide d i s s o l v i n g into the prawn t i s s u e or both. increased  The tissue pHs o f the NMAP and the c o n t r o l prawns  as a r e s u l t  of m i c r o b i a l production  compounds such as ammonia and amines.  o f various  spoilage  These r e s u l t s are i n accordance  with the findings i n the changes i n the TVBN and the TMAN concentrations i n t h i s study. treatments  Since prawn tissue pHs were s i g n i f i c a n t l y d i f f e r e n t among  on every  sampling  day as w e l l as during the whole storage  p e r i o d , i t might be a s u i t a b l e index to help categorize prawns from the three treatments of the same storage p e r i o d .  The d i s c r i m i n a t i n g power of  the t i s s u e pH was evident i n the r e s u l t of the d i s c r i m i n a n t a n a l y s i s .  5.3. Exudate formation The  greatest reduction i n tissue pH occurred  which had the highest volume of exudate.  i n the CMAP prawns  This i s i n general agreement  with several reports by Penny (1977), Khan (1977), Warriss  (1982), and  Warriss and Brown (1987) on porcine muscle.  However, a low c o r r e l a t i o n  between exudate volume and pH was evident  (r=-0.492  r=-0.562 i n T r i a l 2, Tables 12 and 14).  i n Trail  1 and  Developments o f the exudates i n  the NMAP and the c o n t r o l prawns were i n accordance with the changes i n t h e i r t i s s u e pHs.  137  Table 13.  EXUDATE PH TSA TSN TMAN IMP ADP AMP HX HXR K L A B RC RO CC CO CF CT OVERALL  IMP ADP AMP HX HXR K L A B RC RO CC CO CF CT OVERALL  C o r r e l a t i o n matrix from factor analysis on data of T r i a l 1 EXUDATE  PH  TSA  TSN  TMAN  1.000 - o . 492 0. 265 0. 268 0. 431 - 0 . 590 - 0 . 638 - 0 . 700 0. 497 - 0 . 223 0. 652 0. 485 0. 500 0. 584 0. 032 - 0 . 187 - 0 . 228 - 0 . 604 - 0 . 492 - 0 . 759 - 0 . 613  1.,000 0. 480 0.,518 0.,355 - 0 . 148 0. 157 0. 026 0. 192 - 0 . 502 0. 083 0. 170 0. 145 - 0 . 054 0. 293 - 0 . 397 0. 643 0. 135 - 0 . 023 0. 484 0. 312  1..000 0,.985 0..587 -0. .747 -0 .484 -0. .754 0..620 -0. .565 0..721 0..605 0..527 0..502 0..291 -0. ,662 0.,390 -0. .567 -0. .623 -0. ,238 - 0 ..394  1..000 0..637 -0. .764 -0 .477 -0 .753 0..668 -0. .604 0..742 0..644 0..558 0,.507 0..341 -0. .671 0..468 -0. .566 -0. .640 -0. .238 -0. .364  1.000 -0.783 -0.603 -0.647 0.854 -0.812 0.810 0.650 0.646 0.513 0.381 -0.632 0.457 -0.521 -0.594 -0.423 -0.356  IMP  ADP  AMP  HX  HXR  1. 000 0. 806 0. 924 - 0 . 849 0. 754 - 0 . 940 - 0 . 655 - 0 . 659 - 0 . 626 - 0 . 323 0. 658 - 0 . 272 0. 773 0. 765 0. 582 0. 604  1. 000 0. 817 - 0 . 602 0. 568 - 0 . 733 - 0 . 387 - 0 . 417 - 0 . 521 - 0 . 307 0. 362 - 0 . 085 0. 651 0. 637 0. 636 0. 523  1.,000 -0. ,782 0.,533 - 0 . ,925 - 0 . 624 -0. ,630 - 0 . ,665 - 0 . 216 0.,603 - 0 . ,169 0.,850 0. 810 0.,662 0. 703  1..000 -0. .680 0..926 0..662 0..648 0..565 0.,325 - 0 . .666 0..326 - 0 . .709 - 0 . .730 - 0 . .533 - 0 . .546  1.000 -0.640 -0.472 -0.496 -0.372 -0.446 0.554 -0.480 0.385 0.427 0.171 0.170  -  Continued next page  138  Table  K L A B RC RO CC CO CF CT OVERALL  RO CC CO CF CT OVERALL  13  continued..  K  L  A  B  RC  1.000 0.698 0.654 0.622 0.279 -0.666 0.245 -0.825 -0.809 -0.668 -0.673  1.000 0.707 0.634 0.232 -0.566 0.274 -0.492 -0.550 -0.423 -0.412  1.000 0.785 -0.015 -0.677 0.166 -0.623 -0.632 -0.502 -0.618  1.000 -0.069 -0.573 0.040 -0.583 -0.510 -0.411 -0.580  1.000 0.181 0.572 -0.091 -0.287 -0.125 0.337  RO  CC  CO  CF  CT  1.000 -0.237 0.643 0.584 0.222 0.659  1.000 -0.054 -0.229 0.127 0.345  1.000 0.839 0.683 0.846  1.000 0.731 0.721  1.000 0.706  OVERALL OVERALL  1.000  139  Table 14.  Correlation matrix from factor analysis of data of 12 days storage of T r i a l 2  EXUDATE EXUDATE pH TSA TSN SPA SPN TMAN TVBN WSP SSP L a b OVERALL RC RO CC CO CF CT  SPN TMAN TVBN WSP SSP L a b OVERALL RC RO CC CO CF CT  pH  TSA  TSN  SPA  1.000 -0. 562 0.277 0.346 0.403 0.473 0.184 0.318 0.106 0.333 0.400 0.298 0.283 -0. 474 -0. 197 -0. 227 -0. 620 -0. 454 -0. 427 -0. 586  1. .000 0..508 0..458 0..362 0..267 0..355 0..209 0,.604 -0..539 -0..523 -0..414 -0..120 -0..261 -0..387 -0..519 0..226 -0..228 -0..352 0..274  1.000 0.990 0.942 0.909 0.777 0.504 0.811 -0.042 0.070 0.063 0.247 -0.864 -0.756 -0.905 -0.351 -0.854 -0.878 -0.372  1 .000 0 .964 0 .933 0 .739 0 .495 0 .788 -0 .008 0 .115 0 .083 0 .281 -0 .880 -0 .760 -0..898 -0 .401 -0 .856 -0 .897 -0 .403  1.000 0 .969 0 .703 0 .475 0 .763 0 .058 0 .170 0 .133 0 .311 -0 .880 -0 .771 -0 .868 -0 .440 -0 .822 -0 .912 -0 .419  SPN  TMAN  TVBN  WSP  SSP  1.000 0.712 0.597 0.749 0.151 0.182 0.193 0.216 -0. 853 -0. 647 -0. 840 -0. 413 -0. 828 -0.893 -0. 509  1. .000 0..452 0,.599 0..203 0..161 0..275 0..003 -0..705 -0..504 -0..811 -0..172 -0..762 -0..629 -0..525  1.000 0.516 0.135 -0.070 -0.057 -0.227 -0.384 -0.023 -0.409 -0.067 -0.516 -0.521 -0.293  1 .000 -0..411 -0..327 -0 .262 0 .071 -0,.783 -0,.597 -0..823 -0 .418 -0..702 -0..845 -0..321  1.000 0 .735 0 .709 0 .012 0 .043 0 .164 0 .096 0 .122 -0 .099 0 .099 -0 .324  Continued next page  Table  14  ,L  a b OVERALL RC RO CC CO CF CT  RO CC CO CF CT  continued.  L  a  b  OVERALL  RC  1.,000 0.,746 0.387 -0.081 -0.165 0.061 -0.115 -0.140 0.030 -0.224  1,.000 0..154 0..013 0..052 -0..024 0..178 -0..008 0..110 -0,.377  1..000 -0..358 -0 .513 -0,.220 -0..388 -0..282 -0..312 -0..057  1.000 0.747 0.915 0.709 0.950 0.954 0.613  1 .000 0 .675 0 .476 0 .648 0 .711 0 .035  RO  CC  CO  CF  CT  1.000 0.394 0.873 0.890 0.556  1..000 0,,614 0.,619 0.,516  1..000 0..904 0..606  1.000 0.483  1 .000  141  Exudate volume also  appeared to have some c o r r e l a t i o n w i t h the  cooked prawn meat texture score (r=-0.759 i n T r i a l 1 and r=-0.586 i n T r i a l 2, Table 12 and 14).  Moisture content i n the muscle has been  r e l a t e d to the j u i c i n e s s of the meat.  In t h i s study, the CMAP prawns  were found to have s o f t e r or mushier texture than the c o n t r o l and the NMAP prawns.  A s i m i l a r f i n d i n g with f i s h f i l l e t s was also reported by  Coyne (1933).  5.4.  Total  psychrotrophic  bacterial  counts and t o t a l  sulphide-  producing psychrotrophic bacterial counts In t h i s study, the CMAP extended the m i c r o b i a l l a g phase to 4 days. I t was also very e f f e c t i v e i n slowing the m i c r o b i a l growth rate during storage of the prawns and was much more e f f e c t i v e than the NMAP. Since these b a c t e r i a appeared to be able to grow w i t h or without oxygen, the majority of the microbial population present m a t e r i a l prawns must have been f a c u l t a t i v e anaerobes.  However, i t was  u n l i k e l y that these f a c u l t a t i v e anaerobes whould have been Staphylococcus  because these microorganisms  growth temperature i s around 37°C.  i n the raw  E.  coli  or  are pathogens whose optimum  According to Gray et a l . ,  i s also u n l i k e l y that these pathogenic microorganisms  which  (1983), i t grow very  slowly, i f at a l l , at r e f r i g e r a t i o n temperature, w i l l grow under a carbon dioxide enriched-atmosphere According  spp.,  to Dainty  even at abusive temperatures such as 10°C. et  al.,  (1979),  Pseudomonas  spp.,  Aeromonas  and Enterobacteriaceae were capable of growing under low oxygen  tension environment even though growth was not as vigorous as i n the normal atmosphere as the storage continued. i n the NMAP and the CMAP prawns.  This probably was what happened  These b a c t e r i a i n c l u d i n g  Pseudomonas  142  spp.  and Alteromonas  are dominant meat spoilage b a c t e r i a and  putrefaciens  also are sulphide producers (Lee et a l . , 1977).  Since these  sulphide  producers played a very s i g n i f i c a n t r o l e i n the spoilage of the prawns, t h e i r population under aerobic conditions has proved (by the stepwise discriminant a n a l y s i s ) to be an important i n d i c a t o r o f q u a l i t y stages of the prawns. Under the carbon dioxide and the nitrogen storage atmospheres, the psychrotrophic populations were markedly lower than that o f the aerobic c o n t r o l treatment.  I t i s c l e a r that the d i f f e r e n c e i n the psychrotrophic  population between the CMAP and the c o n t r o l treatments  i s due to the  b a c t e r i o s t a t i c e f f e c t of carbon dioxide and the absence o f oxygen. The same explanation may be used f o r the NMAP treatment where oxygen was absent and the carbon dioxide l e v e l during storage up to 13%.  i n the NMAP treatment  increased  This small amount o f carbon dioxide i n the  NMAP bags l i k e l y i n h i b i t e d psychrotrophic growth but not as strongly as i n the CMAP bags. NMAP  treatment  Even though the concentration o f carbon dioxide i n the was  high  enough  to  inhibit  the growth  of the  psychrotrophs, i t was too low to induce a l a g phase.  5.5.  Trimethylamine-nitrogen concentration Dominant  Alteromonas  meat  spoilage  putrefaciens  b a c t e r i a such  as  Pseudomonas  are also TMAO reducers (Lee et al.,  spp.  and  1977). The  r e s u l t s showed that the NMAP system strongly favored the production of TMA.  This i s i n agreement withobservations made by Watson (1939) that  TMA was formed by anaerobic r e s p i r a t i o n of spoilage organisms. CMAP system d i d not favor considered  an  anaerobic  the production environment.  of TMA even though Initital  nhibition  But the it  is  of TMA  143  production i n the CMAP prawns was probably a r e s u l t o f the b a c t e r i o s t a t i c e f f e c t of carbon d i o x i d e . TMAN concentration storage  possibly  microorganisms.  i n the aerobic  because  TMA  was  c o n t r o l prawns dropped catabolized  by  some  during other  Therefore, i n t h i s study, under normal a i r atmosphere,  TMA should be s u i t a b l e as a measure o f prawn q u a l i t y f o r up to 2 weeks.  5.6.  Total v o l a t i l e basic nitrogen concentration The l a r g e s t amount of TVBN was produced i n the c o n t r o l prawns f o l -  lowed by the NMAP and the CMAP prawns r e s p e c t i v e l y .  This i s because the  c o n t r o l treatment, which was a normal atmospheric environment i n a d d i t i o n to  the low temperature  spoilage microorganisms.  of 1°C, strongly  favored the growth  Microorganisms such as Pseudomonas  shown to be capable of producing v o l a t i l e  basic  o f meat  species were  compounds  (Cobb and  Vanderzant, 1971). On the contrary, the CMAP system r e s u l t e d i n the smallest production of v o l a t i l e basic compounds among a l l treatments i n t h i s study. This was mainly the r e s u l t of the i n h i b i t o r y e f f e c t s o f carbon dioxide toward the normal meat-spoilage microorganisms. According to Yeh et al., (1978) and Satake et al., (1952), t i s s u e enzymatic production o f ammonia was evident over a wide pH range from s l i g h t l y a c i d i c  to a l k a l i n e .  Yeh et al.,  (1978) also reported that there were s i g n i f i c a n t amounts of these enzymes (adenosine deaminase and AMP deaminase) i n shrimp muscle. i s speculated here that  Therefore, i t  considerable amounts of v o l a t i l e b a s i c compounds  i n the CMAP prawns were the r e s u l t of the a c t i v i t i e s of n a t u r a l tissue enzymes i n prawns which were s t i l l a c t i v e at s l i g h t l y a c i d i c pH.  144  Cobb and Vanderzant  (1971)  found  that  species were  Pseudomonas  strongly capable o f producing v o l a t i l e basic compounds. this  study  showed that the best c o r r e l a t i o n s  The r e s u l t s i n  o f TVBN were to t o t a l  anaerobic sulphide-producing psychrotrophic b a c t e r i a l count (r=0.597) and scores f o r flavour and f o r odour o f cooked prawn meat (r=-0.521 and r=-0.516 r e s p e c t i v e l y ) .  This f i n d i n g i s also i n general agreement with  Gagnon and F e l l e r (1958).  5.7.  K-value and r e l a t e d  compounds  No ATP was detected i n prawn samples i n T r i a l 1.  I t appears that  ADP degradation was accelerated i n the CMAP prawns but was delayed i n the NMAP prawns. As a consequence, AMP i n the CMAP prawns was expected to be formed more r a p i d l y than i n the other treatments. was  However, since there  no s i g n i f i c a n t difference i n AMP concentration i n prawns from a l l  treatments of each sampling day, t h i s may i n d i c a t e that AMP was degraded more slowly i n the CMAP prawns than i n the NMAP prawns and was degraded most r a p i d l y i n the c o n t r o l prawns. was  formed f a s t e r  As a r e s u l t , IMP i n the NMAP prawns  than i n the CMAP prawns and thus was subjected to  degradation sooner than that i n the CMAP prawns. most r a p i d l y  IMP which was formed  i n the c o n t r o l prawns was degraded f i r s t .  Inosine was  formed accordingly to the IMP decomposition rate and hypoxanthine was also formed accordingly to the inosine decomposition r a t e . The  r e s u l t s i n t h i s study showed high c o r r e l a t i o n between K-value  and IMP (r=-0.940).  This supports the f i n d i n g by E h i r a (1976) that K-  value was influenced most strongly by the IMP decomposition r a t e . this  In  study, the r e s u l t s showed that development of high K-values was  f a s t e r i n the NMAP than i n the CMAP system.  145 5.8.  S a l t - s o l u b l e and WSP c o n c e n t r a t i o n s  As a result of p r o t e o l y s i s , SSP were released from the muscle proteins and then were further hydrolyzed to form WSP. results  showed that proteolysis progressed  control prawns after approximately  In this study, the  most rapidly  i n the aerobic  one week of storage when SSP started  to decrease and WSP gradually increased and then peaked by day 12. In contrast, proteolysis i n the CMAP and the NMAP prawns did not take place extensively u n t i l after about 2 weeks of storage since the concentrations of  the SSP fractions  until  day 16.  This  i n the CMAP and the NMAP prawns d i d not decrease i s because  growth  of microorganisms,  such as  Pseudomonas species which are capable of degrading these proteins (Borton et al.,  1970; Cobb and Vanderzant, 1971), were favored under the normal  a i r atmosphere but were i n h i b i t e d under carbon dioxide atmosphere. At the l a s t period of storage of the aerobic control prawns, there was another increase i n the SSP. degradation  of the major  stromal  This was possibly the r e s u l t of the proteins, collagen. Some Pseudomonas  species were found to produce collagenases  (Ockerman et al.,  1969; Yada  and Skura, 1981). The prawns  maximum concentrations were  much  lower  than  of WSP fractions that  i n the CMAP and NMAP  of the control prawns.  The WSP  fractions i n the CMAP and the NMAP prawns increased at a much slower rate and  peaked after the WSP f r a c t i o n i n the control prawns.  The control  prawns had the highest concentration of WSP; and the NMAP prawns had a slightly  higher  concentration of the WSP than the CMAP prawns.  results are i n agreement with those reported by Borton et al., Ockerman et al.,  These  (1970) and  (1969) that WSP increases with increase i n tissue pH.  146  5.9.  Colour As expected, the NMAP and the CMAP system prevented degradation of  the colour (somewhat enhanced the redness and somewhat lightened the colour) of the raw, headless, shell-on pink prawns as i n d i c a t e d by the Hunter a and L values r e s p e c t i v e l y .  This was because the i n e r t gas,  n i t r o g e n , o f the NMAP system and the anaerobic conditions o f both the NMAP and the CMAP systems provided p r o t e c t i o n from oxygen. Prawns under the nitrogen atmosphere had higher Hunter a values indicating  that  nitrogen  gas provided  better  protection  (for  the  carotenoid pigments against oxidation) than d i d the carbon dioxide i n the CMAP system.  The Hunter L value was higher i n the CMAP prawns than i n  the NMAP prawns.  I t was probably because a c i d i c conditions induced by  the d i s s o l v e d carbon dioxide causing some destruction o f these carotenoid pigments i n the CMAP prawns.  But f o r the NMAP prawns, there appears to  be no explanation f o r t h i s phenomenon. One p o s s i b i l i t y may be the carbon dioxide l e v e l i n the NMAP bags was s u f f i c i e n t to somehow destroy the carotenoid pigments but i t was too low to induce an a c i d i c  condition.  Colour, which i s one of the major q u a l i t y determinants f o r consumers of fresh prawns, when faded, resulted  i n lowered  color  scores  from the  i n colour of the NMAP prawns  increased  sensory panel. Intensity  of yellowness  during the e a r l y stages of storage and l a t e r decreased storage i n both t r i a l s .  a t the end of  However, yellowness i n t e n s i t y of the CMAP prawn  colour increased and reached higher values i n T r i a l 2 than i n T r i a l 1. Moreover, the r e s u l t s showed that changes i n the Hunter b value of a l l prawns i n both t r i a l s were d i f f e r e n t .  This maybe because the raw materi-  147 a l prawns i n T r i a l 2 were more yellow i n colour to begin with or i t might be due to the stage of the prawns during t h e i r spawning c y c l e .  5.10.  Sensory characteristics The NMAP prawns had a very offensive p u t r i d odour and a strong smell  of hydrogen sulphide.  Hydrogen sulphide production was probably the  r e s u l t of"amino a c i d decomposition. preserved  I t was reported that a high pH meat  i n vacuum, when s p o i l e d , produced a strong p u t r i d odour and  hydrogen sulphide (Bern e t a l . , 1976; Taylor and Shaw, 1977; N i c o l et a l . , 1970).  I t i s p o s s i b l e that TMA also made a s i g n i f i c a n t c o n t r i b u t i o n to  the p u t r i d odour.  The odour o f the CMAP prawns was not as offensive as  that of the NMAP prawns. Heating apparently influenced the odour score as the r e s u l t s showed that the scores f o r cooked prawn meat odour were s i g n i f i c a n t l y d i f f e r e n t i n both  trials  (P<0.001) from the scores  f o r raw prawn meat odour.  V o l a t i l e as w e l l as n o n - v o l a t i l e compounds i n prawns might be a l t e r e d by heat i n the cooking  process  to produce strong odours.  For instance,  carotenoid, when subjected to heating, could r e s u l t i n v o l a t i l e compounds such as toluene which has an unpleasant smell (Marty and Berset, 1986). The  results  showed that the scores  f o r cooked prawn meat odour were  generally lower than the scores f o r raw prawn meat odour. The score f o r cooked prawn meat flavour i n T r i a l 1 appeared to have a  considerably  (r=-0.730).  good c o r r e l a t i o n  with  IMP (r=0.765) and hypoxanthine  IMP may have influenced the cooked prawn meat f l a v o u r score  by enhancing prawn flavour and hypoxanthine may influence the score for cooked prawn meat f l a v o u r by imparting the undesirable b i t t e r t a s t e .  148  In  both  trials,  the score  f o r cooked prawn  meat  texture had  considerably good c o r r e l a t i o n with exudate volume (r=-0.759 and r=-0.586 respectively,  Tables  12 and 14) as mentioned e a r l i e r .  The o v e r a l l  sensory score had i t s best c o r r e l a t i o n with the score f o r cooked prawn meat odour and flavour  (r=0.846 and r=0.721 f o r T r i a l  r=0.954 f o r T r i a l 2 r e s p e c t i v e l y , Tables 12 and 14).  1, r=0.950 and Moreover, i t was  also h i g h l y c o r r e l a t e d to a l l m i c r o b i o l o g i c a l v a r i a b l e s i n T r i a l 2. may  This  imply that cooked prawn meat odour and flavour were the greatest  contributors  to the o v e r a l l score and sensory q u a l i t y of the prawns and  were dependent on the m i c r o b i o l o g i c a l q u a l i t y o f the prawns.  5.11.  S h e l f - l i f e of the prawns In t h i s study, the CMAP system f o r prawns increased the m i c r o b i a l  l a g phase to 4 days as w e l l as s i g n i f i c a n t l y decreased psychrotrophic b a c t e r i a l growth rate during the logarithmic shelf-life result  of  phase.  The extension of  o f the prawns obtained by the CMAP system i s p r i m a r i l y a a  failure  of  the contaminating  pseudomonads, to grow s u f f i c i e n t l y  organisms,  to cause spoilage.  increased l a g phase f o r b a c t e r i a i n the NMAP prawns.  probably  There was no  However, m i c r o b i a l  growth i n the NMAP prawns was retarded when compared to that  i n the  c o n t r o l prawns. A p r i n c i p a l factor i s the absence of oxygen and p o s s i b l y the accumulation o f carbon dioxide which has a marked i n h i b i t o r y e f f e c t on pseudomonads. S h e l f - l i f e of prawns i s dependent on the index used.  For T r i a l 2,  based on the t o t a l psychrotrophic b a c t e r i a l count of l o g 1 0 6.0 to 6.5, the s h e l f - l i v e s of the prawns i n t h i s study were 1.5, 7, and 2 days f o r the c o n t r o l , the CMAP, and the NMAP prawns r e s p e c t i v e l y .  Based on the  149  TVBN concentration of up to 50 mg%, s h e l f - l i v e s o f the prawns i n t h i s study were 6 days f o r both the c o n t r o l and the NMAP prawns and 12 days f o r the CMAP prawns.  These findings are generally i n agreement with  Layrisse and Matches (1984).  However, the s h e l f - l i f e of the pink prawns  under carbon dioxide i n t h e i r study was longer. material  prawns  i n this  study  were  This i s because the raw  more h e a v i l y  contaminated  microorganisms than ones used by Layrisse and Matches (1984). the  prawns i n t h i s  laboratory. (Ehira,  with  Moreover,  study were 4 days o l d when they a r r i v e d at the  I f a K-value o f 20% i s used as a l i m i t f o r f i s h  1976), the raw m a t e r i a l  prawns  i n this  study  could  freshness not be  considered fresh since the i n i t i a l K-value were 31.9%. Results from the f a c t o r analysis also showed that a l l m i c r o b i o l o g i c a l v a r i a b l e s and TMAN concentration were the d e s c r i p t o r s of Factor 1. Moreover, pH and SPA can be used to determine or p r e d i c t s h e l f - l i f e or spoilage  of the prawns (as the r e s u l t from the stepwise  analysis).  Determination  discriminant  of pH i s simple, r a p i d , and inexpensive.  It  was also s i g n i f i c a n t l y d i f f e r e n t among a l l treatments on every sampling day as w e l l as during the whole storage. factor  Moreover, i t was an important  i n the effectiveness of carbon dioxide  storage  atmosphere i n  i n h i b i t i n g m i c r o b i a l growth which r e s u l t e d i n extended s h e l f - l i f e of the prawns.  SPA presented d i r e c t information on the population of spoilage  b a c t e r i a that contaminated the prawns. SPA  By p u t t i n g i n the data f o r pH and  of the prawns, a p l o t of two canonical v a r i a b l e s can be obtained.  This p l o t helps as a geometric presentation o f the q u a l i t y o f the prawns w i t h i n the three regions o f q u a l i t y : good, acceptable, or unacceptable.  150  6.  CONCLUSION  151  6.  CONCLUSION  In general, the r e s u l t s i n t h i s study were i n agreement with Cobb et  al.,  (1973) that the s h e l f - l i f e of the prawns was s i g n i f i c a n t l y l i m i t e d  because of the a c t i v i t i e s of the m i c r o b i a l f l o r a ; and to a l e s s e r extent because of the a c t i v i t i e s o f the n a t u r a l t i s s u e enzymes o f the prawns. Carbon dioxide was the only f a c t o r i n the packaging atmospheres that contributed the b a c t e r i c i d a l e f f e c t .  While there was high concentration  of carbon dioxide i n the CMAP bags, a small concentration  of carbon  dioxide d i d develop i n the NMAP bags as a r e s u l t of m i c r o b i a l r e s p i r a t i o n and biochemical r e a c t i o n s . due  Any b e n e f i c i a l e f f e c t of the MAP system was  to (1) e x c l u s i o n of oxygen and (2) the concentration  dioxide present i n the bag.  of carbon  Carbon d i o x i d e , with i t s high s o l u b i l i t y i n  the aqueous phase o f the prawn muscle t i s s u e was responsible f o r the s i g n i f i c a n t pH drop.  The reduction of the t i s s u e pH caused a release of  the sarcoplasm from the muscle.  The CMAP prawns had the lowest t i s s u e pH  and the highest exudate volume. Effectiveness of these storage atmospheres i n i n h i b i t i n g m i c r o b i a l growth appears to r e l a t e atmosphere.  to the amount of carbon dioxide gas i n the  F a c u l t a t i v e anaerobes appeared to be predominant i n the  prawns i n t h i s study since they could grow with or without the presence of oxygen. under  The growth o f these microorganisms was markedly  the CMAP and NMAP system.  producing  were  capable o f  sulphides, TMAO, v o l a t i l e bases, and had strong  proteolytic  activities, relative treatment.  These  organisms  inhibited  the development of TMAN, TVBN, SSP, and WSP occurred i n  to the growth  of these  psychrotrophic  bacteria  i n each  An exception was that TMA production was favored under the  152  anaerobic  conditions  of the NMAP  system.  indicated that these sulphide-producing  These  results  strongly  psychrotrophic bacteria played a  very s i g n i f i c a n t role i n spoilage of the prawns. Development of K-value was  accelerated more by the NMAP than the CMAP as a consequence of the  d i f f e r e n t decomposition rates of ADP as well as AMP i n prawns i n these two  storage  atmospheres.  The development of K-value i n the prawns was  influenced most strongly by IMP decomposition r a t e . As indicated by the Hunter L and a values, colour of the prawns was well preserved under the NMAP and the CMAP systems due to their anaerobic conditions oxygen.  that  protect  the carotenoid  pigments  against  oxidation by  While the a c i d i c condition, induced by dissolved carbon dioxide,  of the CMAP prawns caused destruction of the pigments, there appears to be no explanation for this phenomenon i n the NMAP prawns except that the carbon dioxide l e v e l i n the NMAP bags was s u f f i c i e n t to somehow destroy the carotenoid pigments i n the prawn s h e l l but i t was too low to induce an a c i d i c condition i n the t i s s u e .  These anaerobic conditions  together  with the presence of carbon dioxide resulted i n the higher redness i n the NMAP prawns than i n the CMAP prawns and the l i g h t e r colour of the CMAP prawns compared to the NMAP prawns. Raw sensory sulphide  and cooked prawn meat odours were the main contributors to the quality of the prawns.  Offensive  p u t r i d odour  smell were c h a r a c t e r i s t i c s of the NMAP prawns.  and hydrogen Softening of  texture was found i n the CMAP prawns i n this study. The  CMAP system extended s h e l f - l i f e of the prawns to about 3 times  that of the c o n t r o l , based on the t o t a l psychrotrophic b a c t e r i a l count or to 2 times based on TVBN concentration.  In contrast to the CMAP system,  the NMAP system did not extend s h e l f - l i f e of the prawns and also resulted  153  i n very strong undesirable sulphide odours.  However, i t was  superior to  the CMAP system i n maintaining the desirable red colour of the prawns. 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Relation between the number of trimethylamineoxide reducing psychrotrophic bacteria and their a c t i v i t y . B u l l e t i n of the Japanese Society of S c i e n t i f i c Fisheries 39:511-518. Sasajima, M. 1974. Studies on psychrotolerant bacteria i n f i s h and s h e l l f i s h . V. The growth or v i a b i l i t y of trimethylamineoxide reducing psychrotrophic bacteria and their a c t i v i t y at subzero temperatures. B u l l e t i n of the Japanese Society of S c i e n t i f i c Fisheries 40:625-630. Schmidt, P.J. and D.R. I d l e r . 1958. Predicting the colour of canned sockeye salmon from the colour of the raw f l e s h . Food Technology 12(1)44-48. Smith, C.E. 1987. F e a s i b i l i t y of Modified Atmosphere Packaging of Fish: A Review. Saskatchewan Reserch Council. Publication number I-4202-1-E-87.  161  S p i n e l l i , J . and J.A. Dassow. 1982. "Fish p r o t e i n s : Their m o d i f i c a t i o n and p o t e n t i a l uses i n the food industry." 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Residual e f f e c t of storage i n an elevated carbon dioxide atmosphere on the m i c r o b i a l f l o r a of rock cod (Sebastes spp.). Applied Environmental Microbiology 52:727-732. Warriss, P.D. 1982. The r a l a t i o n s h i p between pH45 and d r i p i n p i g muscle. Journal of Food Technology 17:573-578. Warriss, P.D. and S.N. Brown. 1987. The r e l a t i o n s h i p s between i n i t i a l r e f l e c t a n c e , and exudation i n p i g muscle. Meat Science 20:65-74.  pH,  Watson, D.W. 1939. Studies of f i s h spoilage IV. The b a c t e r i a l reduction of trimethylamine oxide. Journal of Fishery Research Board of Canada 4:252-266.  162  Watts, D.A. and W.D. Brown. 1982. Histamine formation i n abusively stored p a c i f i c mackerel: e f f e c t of C0 2 -modified atmosphere. Journal of Food Science 47:1386-1387. Wilkinson, L. 1988. SYSTAT: The System for S t a t i s t i c s . Computer software. SYSTAT Inc., 1800 Sherman Avenue, Evanston, I l l i n o i s . Wolfe, S.K. 1980. Use of CO- and C0 2 -enriched atmospheres for meat, f i s h , and produce. Food Technology 34(3)55-58,63. Woyewoda, A.D., S.J. Shaw, P.J. Ke, and B.G. Burns. 1986. Recommended Laboratory Methods f o r Assessment of Fish Quality. Canadian Technical Report of Fisheries and Aquatic Sciences. No.1448, p.113. Yada, R.Y. and B.J. Skura. 1981. Some biochemical changes i n sarcoplasmic depleted intact beef muscle inoculated with Pseudomonas fragi. Journal of Food Science 46:1766-1773,1776. Yeh, C.S., R. Nickelson I I , and G. Finne. 1978. Ammonia-producing enzymes i n white shrimp t a i l s . Journal of Food Science 43:1400-1401,1404.  APPENDIX A:  CHLORAMPHENICOL TREATMENT  164  A-l.  Chloramphenicol Chloramphenicol i s an aromatic a n t i b i o t i c o r i g i n a l l y i s o l a t e d from  Streptomyces  venezualae  (AHFS Drug Information 1986,  Section 8:12.08).  I t i s a benzene compound and i s c l a s s i f i e d into the group of a n t i b i o t i c code number 8.1.2.1.1. by bacteriostatic.  Berdy  Nevertheless,  (1974).  Chloramphenicol  it  be  can  i s usually  bactericidal  concentrations or against h i g h l y susceptible organisms.  at  high  Chloramphenicol  i n h i b i t s p r o t e i n synthesis i n susceptible microorganisms  by binding to  the 50S ribosome subunits. As a r e s u l t , peptide formation i s i n h i b i t e d . Chloramphenicol i s active against various b a c t e r i a but i n a c t i v e against fungi. A-2.  Purpose The  purpose  microorganisms which  of using chloramphenicol was  to  inhibit  growth  of  i n the prawns hence e l i m i n a t i n g the m i c r o b i a l a c t i v i t i e s  degrade the prawns.  Therefore, only the degradation caused  by  prawn t i s s u e enzymes could be examined. A-3.  Concentration of the chloramphenicol solution Many  experiments  concentration  of  the  were  carried  out  chloramphenicol  to  determine  solution  and  the the  working treatment  procedure. F i r s t , 50 gm headless shell-on prawns were soaked i n 500 ml of (1) deionized d i s t i l l e d water as the c o n t r o l ,  (2) 100 ppm  chloramphenicol  s o l u t i o n , and (3) 200 ppm chloramphenicol s o l u t i o n f o r 10 minutes at room temperature.  A f t e r that the prawns were removed and the t o t a l  counts were determined  using t r y p t i c a s e  soy ager and  plate  21 °C incubation  165  temperature.  The enumeration was done a f t e r 48 hours of incubation.  The  r e s u l t s were as follows: Total p l a t e counts  Treatments Control  6.10xl0  100 ppm  1.14xl0  200 ppm  8.60xl0  5  6  5  cfu/gm cfu/gm cfu/gm  Second, 40 gm headless s h e l l - o n prawn were soaked i n 500 ml of (1) deionized d i s t i l l e d water as the c o n t r o l and (2) 100 ppm chloramphenicol solution  f o r 30  minutes  at room  temperature.  The  discarded and each prawn sample was packed i n a s t e r i l e stored at 4°C f o r 24 hours.  solutions  were  p l a s t i c bag and  The t o t a l p l a t e counts were determined. The  r e s u l t s were as follows: Treatments  Total p l a t e counts  Control  6.10xl0  100 ppm  3.81xl0  5  5  cfu/gm cfu/gm  T h i r d , 50 gm headless s h e l l - o n prawns were soaked i n 500 ml of deionized d i s t i l l e d water as the c o n t r o l and (2) 100 ppm chloramphenicol s o l u t i o n f o r 24 hours at 4°C.  The t o t a l p l a t e counts were determined.  The r e s u l t s were as f o l l o w s : Treatments  Total p l a t e counts  Control  6.10xl0  100 ppm  4.46xl0  5  5  cfu/gm cfu/gm  Fourth, 50 gm headless s h e l l - o n prawns were submerged i n 1000 ml of (1) deionized d i s t i l l e d water as the c o n t r o l , (2) 100 ppm chloramphenicol  166  solution,  (3)  200  ppm  chloramphenicol  chloramphenicol s o l u t i o n . the  solutions  solution,  and  (4)  400  ppm  Each was shaken under vacuum s u c t i o n to allow  to penetrate  into  soaking f o r 12 hours at 4°C.  the undershell areas  and  then  left  A f t e r soaking, the s o l u t i o n s were removed  and each prawn sample was packed i n a s t e r i l e p l a s t i c bag, and stored f o r 4 days at 4°C.  The t o t a l plate counts were determined.  The r e s u l t s were  as shown i n Table A - l . These r e s u l t s revealed that 200 ppm  chloramphenicol s o l u t i o n with  the treatment procedure as described i n the fourth experiment showed some inhibition  as  the  chloramphenicol microbial  microbial  solution  count  count  decreased  concentration  remained  slightly.  increased  constant.  This  to  means  When  the  400  ppm,  the  that  400  ppm  chloramphenicol s o l u t i o n s o l u t i o n e f f e c t i v e l y stopped the growth of the microorganisms present i n the prawn sample.  Therefore, i t can be used to  serve the purpose. A-4.  Effect on the K-value of the prawns There was  a concern  as to whether t h i s  selected  chloramphenicol  s o l u t i o n treatment would a f f e c t the nucleotides w i t h i n the prawn t i s s u e i n any  way.  An  choramphenicol  experiment  was  set up  to examine the e f f e c t of  the  treatment.  Twenty-five  grams  treatment (Table A - l ) .  of  headless  shell-on  prawns  were  used  per  A f t e r the treatments, the muscle was blended  extracted f o r K-value determination as decribed i n s e c t i o n 3.5.5.  and  Prior  to i n j e c t i o n onto the HPLC column, 2 ml of the extract was mixed with 100  pil of the  internal  standard 5-bromouracil  stock s o l u t i o n .  d i l u t i o n process was c a r r i e d out with the soaking s o l u t i o n s .  The  same  Table A - l .  Total aerobic psychrotrophic counts at 21°C 48 hours of fresh prawn samples subjected to control and chloramphenicol treatments  Counts Zero = Counts a f t e r the soaking Counts F i n a l = Counts a f t e r the whole treatment  cfu/gm Counts Zero  Control  4.53xl0  Chloramphenicol 100 ppm  5.00xl0  Chloramphenicol 200 ppm  5.33xl0  Chloramphenicol 400 ppm  8.10xl0  3 2 3 2  Counts F i n a l  2.20xl0 3.41xl0 4.20xl0 8.lOxlO  7 4 2 2  168  The K-values obtained from t h i s experiment are shown i n Table A-2. I t appeared that the i n i t i a l q u a l i t y of the prawn used i n t h i s experiment was poor and the chloramphenicol treatment sample had the lowest K-value among a l l the treated samples. From the r e s u l t s , i t appeared  that the treatment procedure alone  lowered the freshness as much as 27% K-value. to  washing  effect  chloramphenicol  of  alone  the  reduced  treatment.  However, t h i s may be due It  also  appeared  that  the freshness as much as 16% K-value  compared to the c o n t r o l treatment but "preserved" the freshness as much as 11% K-value compared to water treatment alone. K-values of both solutions appeared to be very s i m i l a r .  I t was pos-  s i b l e that the ATP and i t s r e l a t e d degraded compounds might have d i s solved into the s o l u t i o n  initially.  The d e c i s i o n was then made that the chloramphenicol should not be used mainly because the whole treatment procedure alone decreased K-value by 27%. K-value.  I t was not conclusive whether or how chloramphenicol a f f e c t s the Furthermore,  i t might cause  organisms i n c l u d i n g fungi i n the samples.  overgrowth  of non-susceptible  169  Table A-2.  The concentrations of ATP and i t s related compounds i n prawn tissue extracts and soaking solutions  Treatment 1 = Prawns without any treatments. Treatment 2 = 25 gm headless shell-on prawns were shaken for 2 minutes shaken under vacuum suction i n 500 ml water and l e t soak f o r 12 hours at 4°C, then drained. Treated prawns were packed i n a s t e r i l e p l a s t i c bag f o r 4 days at 4°C. Treatment 3 = 25 gm headless shell-on prawns were shaken f o r 2 minutes shaken under vacuum suction i n 500 ml of 400 ppm chloramphenicol solution and soaked f o r 12 hours at 4°C, then drained. Treated prawns were packed i n a s t e r i l e p l a s t i c bag f o r 4 days at 4°C. Treatment 4 = 25 gm headless shell-dn prawns were shaken f o r 2 minutes shaken under vacuum suction i n 500 ml water, then drained. Prawns were packed i n a s t e r i l e p l a s t i c bag f o r 4 days at 4°C. Solution 1 = The water drained out from treatment 2. Solution 2 = The chloramphenicol solution drained out from treatment 3.  Millimoles/25gm HLSO prawn  IMP  ATP  ADP  AMP  Hx  HxR  % K value  1  19..538  0,.000  2..150  4..292  48,.814  7,.942  68..599  2  2..347  0..000  1..485  0.,500  89..811  5..280  95.,643  3  7..029  0,.000  1..528  0..000  39..432  6,.203  84..210  4  13..967  0..000  3..145  0.,911  122..738  9..973  88..043  HxR  % K value  Extract  Millimoles/25gm HLSO prawn Soaking solution  IMP  ATP  ADP  AMP  1  24.776  0.000  5.468  0.000  615.897  0.000  95.319  2  30.822  0.000  6.762  0.000  517.910  14.631  93.408  Hx  APPENDIX B: SAMPLE MEANS AND STANDARD DEVIATIONS  Table B - l .  T r i a l 1:  Sample means and standard deviations  (n=6) of carbon dioxide, oxygen, and nitrogen  concentrations  (%) i n the headspace atmospheres of CMAP bags on indicated sampling days  DAY  C02  02  N2  0  93,.11±1..98  1.,15±0..59  5..74±1..47  3  81. ,01±5..19  2..22+0. .88  16..77+4. .32  9  88,.09±2.,69  0..16±0..04  .66 11..74+2.  18  88,.30+4. .56  0.,11±0..03  11.,59±4..53  Table B-2.  T r i a l 1:  Sample means and standard deviations  (n=6) of carbon dioxide, oxygen, and nitrogen  concentrations  (%) i n the headspace atmospheres of NMAP bags on indicated sampling days  DAY  C02  02  N2  0  0..56±0..15  1.02±0..11  98,.42±0..15  3  2..09+0..25  0.,70±0..88  97.,21±0..71  9  3.32±0.,18  0.,12+0.,04  96..56±0..16  18  9..04±0..44  0.,16±0.,05  90..80±0..46  173  Table B-3. T r i a l 1:  Sample means and standard deviations  (n=15) of pHs of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  7.18±0.10  7.18±0.10  7.1810.10  3  7.22±0.00  6.62±0.10  7.1010.10  6  7.34±0.10  6.6110.00  7.1410.10  9  6.65±0.00  7.3510.10  12  6.7910.10  7.3710.00  15  6.84+0.00  7.3310.10  18  6.8910.10  7.3410.00  Table B-4.  T r i a l 1: Sample means and standard deviations  (n=3) of exudate volumes (ml) of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  0.00±0.00  0.0010.00  0.0010.00  3  2.5710.70  7.4711.50  4.5710.70  6  3.3012.10  7.2011.80  5.1011.50  9  8.1712.50  3.5011.30  12  7.3311.50  6.4712.30  15  9.9311.80  7.1710.90  18  12.0011.50  5.2711.60  175  Table B-5. (n=6)  T r i a l 1:  Sample means and standard deviations  of t o t a l aerobic psychrotrophic b a c t e r i a l counts  (Logi 0 cfu/gm) of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  5.71±0.10  5.71±0.10  5.71±0.10  3  7.1810.20  5.7410.10  6.7510.20  6  8.5910.10  6.1110.10  7.3210.10  9  6.6510.30  7.6710.10  12  7.0010.10  7.4310.00  15  7.0710.10  7.7810.30  18  7.2310.30  7.81+0.10  Table B-6. T r i a l 1:  Sample means and standard deviations  (n=6) of t o t a l anaerobic psychrotrophic b a c t e r i a l counts (Log 1 0 cfu/gm) of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  5.80±0.10  5.80±0.10  5.80±0.10  3  7.1L+0.10  5.70±0.10  6.7L+0.10  6  8.4610.20  6.08±0.10  7.30+0.10  9  6.77±0.10  7.70±0.10  12  6.96±0.10  7.84+0.10  15  7.04+0.20  7.89±0.10  18  7.23±0.30  7.83±0.20  Table B-7.  T r i a l 1: Sample means and standard deviations  (n=6) o f TMAN concentrations (gm/lOOgm t i s s u e ) o f the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  0.50±0.03  0.5010.03  0.50±0.03  3  0.9010.51  0.4110.18  2.6212.77  6  6.9911.52  4.4814.29  9.0712.57  9  5.48+4.82  30.3716.05  12  21.4415.56  40.60+3.58  15  26.2018.13  58.2015.44  18  41.6910.62  65.0112.63  178  Table B-8.  T r i a l 1:  concentrations  Sample means and standard deviations (n=6) of ADP  (nanomoles/ml extract) of the c o n t r o l , the CMAP, and the  NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  7392.17±2569.76  7392.17±2569.76  7392.1712569.76  3  4717.051 471.86  4028.491  6470.581 936.84  6  4041.4311000.39  3486.981 811.70  5621.2211453.77  9  2780.831 966.31  4006.561 639.53  12  3284.111 721.70  824.5111409.06  15  3005.1211014.48  2218.4811619.36  18  2792.561 660.12  1899.461 270.66  71.48  179  Table B-9.  T r i a l 1:  concentrations  Sample means and standard deviations (n=6) of AMP  (nanomoles/ml extract) of the c o n t r o l , the CMAP, and the  NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  37303.31±5661.37  37303.31±5661.37  37303.31±5661.37  3  18093.41±1608.86  23119.19±6344.96  22772.34±6056.19  6  5614.33± 662.49  7789.81± 111.27  8162.80±1764.03  9  5668.90±1504.82  4003.93± 700.42  12  2910.35± 433.05  3249.40±1562.68  15  2057.23±1408.41  1679.44± 789.79  18  1747.69± 665.70  1368.69± 492.82  180  Table B-10.  T r i a l 1:  Sample means and standard deviations (n=6) of IMP  concentrations (nanomoles/ml extract) of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  51516.30±13281.76  51516.30±13281.76  51516.30±13281.76  3  35131.96± 3832.63  39742.30±11934.51  43987.30±  265.87  6  15671.61± 7949.21  27810.78± 5994.80  21736.31±  685.88  9  18957.11+ 4697.93  2990.83± 2632.64  12  15824.26+ 4209.94  7826.04± 6801.99  15  9516.56± 7286.93  0.00±  0.00  18  6535.33± 5764.07  0.00+  0.00  181  Table B - l l .  T r i a l 1:  Sample means and standard deviations (n=6) of  inosine concentrations (nanomoles/ml extract) of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  37548.35±9743.14  37548.35± 9743.14  37548.35±9743.14  3  34763.81±2843.56  38614.07± 4654.66  44297.14±8374.08  6  27126.19+ 816.16  41444.89±11352.35  37769.53±3214.53  44311.13± 3397.51  14768.67±4609.46  12  31738.83±21514.73  12162.87+7014.71  15  26189.95+14771.40  3097.89±3593.48  18  20212.61± 6177.85  1896.95±3285.61  9  182 Table B-12.  T r i a l 1:  Sample means and standard deviations (n=6) of  hypoxanthine concentrations (nanomoles/ml extract) of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  8086.95±3239.63  8086.95± 3239.63  8086.95± 3239.63  3  13115.36±7349.20  18991.42± 5115.77  11563.57± 5633.67  6  35991.61+9966.30  43859.93±19196.37  45284.63± 9467.29  9  52262.38124321.56  101734.42±32913.38  12  76221.611 6610.78  103246.361 8057.00  15  75252.02120470.06  108282.471 5248.81  18  85519.191 3367.70  105050.68110211.14  183  Table B-13.  T r i a l 1:  Sample means and standard deviations  (n=6) of K-values (%) of the control, the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  31.92±1.81  31.92±1.81  31.92±1.81  3  45.01±6.28  46.62±2.65  42.94±2.74  6  71.66±9.02  68.36±3.86  68.37±3.25  9  77.30±7.16  91.28±3.66  12  83.03±1.86  89.08±6.61  15  88.44±6.44  96.68±1.92  18  90.98±5.13  97.03±0.36  184  Table B-14.  T r i a l 1:  Sample means and standard deviations  (n=30) of Hunter L values of the control, the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  37.77±0.53  37.77±0.53  37.7710.53  3  37.82±0.38  39.6110.65  39.5410.65  6  37.74+0.80  40.5311.43  40.0110.14  9  40.55+0.34  40.1510.82  12  41.20+0.24  40.2611.02  15  41.29+0.95  41.15+0.67  18  41.5710.52  41.4510.68  185  Table B-15.  T r i a l 1:  Sample means and standard deviations  (n=30) of Hunter a values of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  9.4710.37  9.47±0.37  9.4710.37  3  9.8010.58  10.7611.99  11.9310.47  6  9.3010.35  11.9711.27  13.1410.25  9  11.6210.22  15.5910.56  12  12.46+0.54  15.2810.80  15  12.8310.63  15.4011.09  18  12.65+0.54  15.5011.19  Table B-16.  T r i a l 1:  Sample means and standard deviations  (n=30) of Hunter b values of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  10.27±0.36  10.2710.36  10.2710.36  3  11.08+0.17  10.9110.48  11.3910.52  6  11.2010.23  11.4010.49  11.7110.32  9  10.6410.18  12.1010.12  12  11.5010.01  11.8510.09  15  11.4810.11  11.7610.45  18  11.8710.20  11.6410.54  187  Table B-17. T r i a l 1:  Sample means and standard deviations  (3 bags/sample and 7 judges) of scores for colour of raw prawn meat of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  5.86±1.88  5.86±1.88  5.86±1.88  3  7.00±1.41  6.14±2.03  5.14±1.96  6  6.00+1.93  5.14±2.10  5.29±1.58  9  6.43±1.29  6.00±2.20  12  5.43±1.76  6.86±0.64  15  7.14±1.12  7.29±0.88  18  5.00±1.77  6.57±1.29  Table B-18. T r i a l 1:  Sample means and standard deviations  (3 bags/sample and 7 judges) of scores f o r odour of raw prawn meat of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  7.14±1.64  7.14±1.64  7.14+1.64  3  7.2911.48  7.1411.64  6.4312.13  6  5.8611.64  6.29+1.67  5.5711.59  9  6.8611.46  4.5711.76  12  6.2911.48  5.7111.91  15  7.2911.39  6.2911.67  18  5.5711.50  4.7112.60  Table B-19. T r i a l 1:  Sample means and standard deviations  (3 bags/sample and 7 judges) of scores f o r colour of cooked prawn meat of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  5.86±2.53  5.86±2.53  5.8612.53  3  6.14±2.17  4.00±1.77  5.8611.96  6  6.00+1.93  5.57+2.13  6.00+2.14  9  5.86+2.10  5.4312.32  12  4.29±2.25  7.8610.83  15  5.71±1.91  7.4310.49  18  4.14±2.36  7.5710.90  190  Table B-20. T r i a l 1:  Sample means and standard deviations  (3 bags/sample and 7 judges) of scores f o r odour of cooked prawn meat of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  6.71±1.48  6.7111.48  6.7111.48  3  5.86±1.96  4.8611.46  6.8611.96  6  4.71+1.91  4.1411.81  3.5711.76  9  4.5711.99  4.0012.20  12  4.14+2.59  4.00+2.27  15  4.2912.05  5.1412.29  18  4.4311.76  3.7111.39  Table B-21. T r i a l 1:  Sample means and standard deviations  (3 bags/sample and 7 judges) of scores f o r flavour of cooked prawn meat of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  6.86±0.64  6.86±0.64  6.86±0.64  3  5.71±1.16  6.43±1.68  6.71±1.75  6  5.86±1.81  6.00±1.07  5.4311.92  9  5.00±2.14  5.14±1.96  12  4.57±1.76  4.86±2.23  15  5.14±2.36  6.14±2.10  18  5.57±1.40  4.29±1.58  192  Table B-22.  T r i a l 1:  Sample means and standard deviations  (3 bags/sample and 7 judges) of scores f o r texture of cooked prawn meat of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  7.4310.73  7.4310.73  7.43+0.73  3  7.2910.88  6.7111.67  7.4311.50  6  7.1410.64  6.7110.70  6.7111.58  9  5.4312.32  7.0011.31  12  6.2911.58  6.7111.67  15  5.8612.17  7.0011.41  18  5.8611.25  6.0011.69  193  Table B-23. T r i a l 1:  Sample means and standard deviations  (3 bags/sample and 7 judges) of o v e r a l l sensory scores of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  6.64±1.00  6.6411.00  6.64+1.00  3  6.55±0.95  5.8811.22  6.4011.39  6  5.93+1.04  5.6410.91  5.4311.11  9  5.6911.13  5.3611.04  12  5.1711.05  6.0011.02  15  5.9011.08  6.5510.72  18  5.1010.96  5.4811.05  Table B-24.  T r i a l 2:  Sample means and standard deviations  (n=6) of carbon dioxide, oxygen, and nitrogen concentrations (%) i n the headspace atmospheres of CMAP bags on indicated sampling days  DAY  C02  02  N2  0  94.57±1.84  1.04±0.36  4.39±1.48  8  84.91±5.47  0.46±0.51  14.63±5.43  16  83.83±1.96  0.30±0.02  15.87±1.95  28  82.20±1.34  0.40±0.08  17.41±1.36  Table B-25. T r i a l 2:  Sample means and standard deviations  (n=6) of carbon dioxide, oxygen, and nitrogen  concentrations  (%) i n the headspace atmosphere of NMAP bags on indicated sampling days  DAY  C02  02  N2  0  0.,95±0..06  1.,63±0..60  97..42±0..65  8  8..86+7. .23  0..10+0. .02  91..04+7. .22  16  6..67±0..33  0..12±0..04  93..21+0. .36  28  12..95±3..93  0..51+0. .50  86,.54±4..42  Table B-26.  T r i a l 2:  Sample means and standard deviations  (n=15) of pHs of the control, the CMAP, and the NMAP prawns on indicated sampling days  Day  CONTROL  CMAP  NMAP  0  7.4510.14  7.4510.14  7.4510.14  4  7.2910.05  6.45+0.05  7.10+0.04  8  7.7610.12  6.5710.07  7.36+0.05  12  8.0910.04  6.8310.27  7.4810.03  16  8.29+0.08  6.8410.05  7.53+0.11  20  8.0010.51  6.8210.35  7.2710.03  24  8.4010.05  6.7110.00  7.3510.12  28  8.4210.04  6.8210.05  7.3410.01  Table B-27. T r i a l 2:  Sample means and standard deviations  (n=3) of exudate volumes (ml) of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  0..00±0..00  0..00±0,.00  0..00±0..00  4  6.,37±1..42  8..17±2..47  6..90±1..47  8  4.,27±0..60  9..93+1. .72  3.,97±2..15  12  5..43+0..47  7.,77±5..35  4..80±1..18  16  3..93+0..83  5..87±1..70  5..97±3..56  20  5..73±1..85  6..03+2. .99  8.,17±1..04  24  11..30±1..39  10.,27±2..27  7.,03±1..76  28  13.,90±3..18  10.,60±6..16  6..5311..77  198  Table B-28. T r i a l 2:  Sample means and standard deviations  (n=6) of t o t a l aerobic psychrotrophic b a c t e r i a l counts (Log 1 0 cfu/gm) of the control, the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  5,,88±0..57  5..8810..57  5..88±0..57  4  7.,63±0..21  5..84±0..41  7.,44±0..24  8  8,.51±0..06  6,,61±0..54  7..90±0..13  12  9..95±0..12  8..10±0..25  9.,23±0..08  16  9..35±0..08  7..42±0..59  7.,97±0..10  20  10..00+0..00  8..94±0..86  11.,65±0..00  9.,29±0..04  8..08±0..06  8..18±0..06  24 28  199  Table B-29. T r i a l 2: Sample means and standard deviations (n=6) of t o t a l anaerobic psychrotrophic b a c t e r i a l counts (Logi 0 cfu/gm) of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  5.,69±0..61  5.,69±0,.61  5..69±0..61  4  7.,71±0..23  5..89±0,.40  7,,50±0..25  8  8..5910..05  6.,85±0..58  7,,97±0..09  12  9.,89±0..06  8..14±0..19  8.,91±0..05  16  9..34±0..07  7..60±0..67  7..92±0..12  20  9.,65±0..26  24 28  9.,33±0.,01  8.,14±0..10  8..23+0.,06  200  Table B-30.  T r i a l 2:  Sample means and standard deviations  (n=6) of t o t a l aerobic sulphide-producing psychrotrophic b a c t e r i a l counts (Log^ cfu/gm) of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  1. ,78±0..35  1. .78±0..35  1. ,78±0.,35  4  4.23±0.,03  2..43±0..49  4.,20±0.,07  8  4..98±0..11  3.,31±0..03  5..1110..02  12  6.,77±0..19  5.,26±0..12  4.,99±0..14  16  4.,85±0..21  4..60±0..63  5..11±0.,12  20  6..19+1,.12  24 28  5..41±0..00  5.,29±0..09  ±0..06  Table B-31.  T r i a l 2:  Sample means and standard deviations  (n=6) of t o t a l anaerobic sulphide-producing psychrotrophic b a c t e r i a l counts (Log 1 0 cfu/gm) of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  0  0..76±0..32  0.,76±0.,32  0.,76±0..32  4  4..12±0..08  2.,38±0.,87  4.,27±0.,20  8  4,,09±0..09  2.68±0..50  4..91±0..22  12  6..58±0..32  4.,63±0.,08  5..22±0..21  16  5.,12±0..17  4.,30±0.,76  4.,92±0..13  20  NMAP  6.,19±1..12  24 28  4,.40±0..00  5.30±0..09  5.,22±0..13  Table B-32.  T r i a l 2:  Sample means and standard deviations  (n=6) of TMAN concentrations (gm/lOOgm tissue) of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  0..06+ 0..01  0.,06± 0..01  0.,06± 0..01  4  2.. 50± 0..78  2..12+ 0..86  3.,48± 0..70  8  12.,50± 0..95  3..68+ 2..00  24.,34± 9..45  12  27,.02± 2..84  15..26+ 9,.78  43..70± 8..15  16  33,.94± 1. .15  31.,66± 6,.02  48.,56± 7,.88  20  22,,20± 8..00  37.,46±10..20  58.,04± 6..80  24  35,.70± 2..08  55.,80± 3..25  74.,00±15..00  28  46,.40±14..66  64,.00111,.15  86..94±24 .07  Table B-33. T r i a l 2:  Sample means and standard deviations  (n=6) of TVBN concentrations (gm/lOOgm tissue) of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  8..65±0..12  8.,65±0..12  8..65±0..12  4  21..67±0..15  16..94±0..11  18.,90±0.,06  8  62..44+0..20  31..92±0..14  64..82+0.,14  12  95.,06±0,.10  47.,46±0..15  103,,60±0..21  16  155,.40±0..09  98..70±0..30  116,,20±0.,16  20  206.,92±0..65  106.,96±0..98  181,.02±0..68  24  226..38±0..47  104..16±0..45  128..80±0..32  28  227,.92±0,.56  109,,06±0..16  116,.06±0..40  204  Table B-34. T r i a l 2: Sample means and standard deviations (n=6) o f water-soluble p r o t e i n concentrations (gm/lOOgm tissue) o f the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0 .  2.77±0.21  2.77±0.21  2.77±0.21  4  3.8210.15  3.15±0.23  4.39±0.39  8  4.4L+0.15  2.85±0.21  3.9L+0.34  12  10.39±0.20  3.94±0.59  5.56±0.44  16  9.83+2.21  7.39±0.67  7.96±0.59  24  4.48+0.53  4.05+0.20  6.41±0.95  28  3.52±1.03  3.56±1.57  2.46±0.35  20  205  Table B-35. T r i a l 2:  Sample means and standard deviations  (n=6) of salt-soluble protein concentrations (gm/lOOgm tissue) of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  3..92+0..49  3..9210..49  3..8210..49  4  4..49+0,.29  5..46±0..13  5..1310..40  8  4..37+0..33  5.,55±0,.32  5..6910..55  12  2.,59±0..44  5..4810..45  5..6910..55  16  2..84±0..72  5..8810..20  5..7110..72  24  5..86±0..83  5..1210..14  4..1910..94  28  0..05+2..44  2..6510..50  2..6911..07  20  206  Table B-36.  T r i a l 2:  Sample means and standard deviations  (n=30) of Hunter L values of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  37.4110.43  37.41±0.43  37.4110.43  4  38.89+2.06  38.6111.76  38.8910.86  8  38.1210.15  40.1910.86  39.8110.40  12  35.7910.26  42.0611.39  39.8910.42  207  Table B-37.  T r i a l 2:  Sample means and standard deviations  (n=30) of Hunter a values of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  12.07±0.43  12.0710.43  12.07+0.43  4  11.6611.42  12.6310.62  13.9410.42  8  11.46+0.48  13.83+1.43  15.3010.94  12  10.8410.71  14.0910.34  14.1610.21  Table B-38.  T r i a l 2:  Sample means and standard deviations  (n=30) of Hunter b values of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  12.03±0.30  12.03±0.30  12.03±0.30  4  12.07±0.78  12.09±0.51  12.41±0.88  8  13.16±0.33  12.8610.41  12.11±0.34  12  12.42±0.53  13.1510.74  12.0510.48  Table B-39.  Trial  2:  S a m p l e means a n d s t a n d a r d  (3 b a g s / s a m p l e a n d 7 j u d g e s ) p r a w n meat o f  the c o n t r o l ,  deviations  of scores for colour of  t h e CMAP, a n d t h e NMAP p r a w n s  i n d i c a t e d s a m p l i n g days  DAY  raw  CONTROL  CMAP  NMAP  0  6.33±1.60  6.33±1.60  6.33±1.60  4  6.17±2.34  7.1710.90  6.0011.00  8  4.5011.61  5.8311.34  6.0011.29  12  4.1711.67  3.8310.90  5.4011.50  on  Table B-40.  T r i a l 2: Sample means and standard deviations  (3 bags/sample and 7 judges) of scores for odour of raw prawn meat of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  7.33±1.25  7.33±1.25  7.33±1.25  4  6.67±1.80  7.00±0.58  6.00±1.63  8  4.00±1.91  5.83±1.77  4.67±0.75  12  2.33±1.11  6.00±1.73  3.50±1.71  211  Table B-41. T r i a l 2:  Sample means and standard deviations  (3 bags/sample and 7 judges) of scores for colour of cooked prawn meat of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  6..67+1..80  6..67±1..80  6..67+1..80  4  5..67±1..89  4..50±1..50  6..83+1..86  8  5.,33±2..05  4..17±2..11  6..67±0..75  12  3.,83±2..19  4.,50±3..55 -  6.,00±1..41  Table B-42. Trial 2: Sample means and standard deviations (3 bags/sample and 7 judges) of scores for odour of cooked prawn meat of the control, the CMAP, and the NMAP prawns on  indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  5.33±1.49  5.33±1.49  5.33±1.49  4  4.0012.38  3.8312.03  4.5012.36  8  2.8311.34  4.0011.67  4.1712.27  12  2.3311.25  3.5012.29  2.1711.34  Table B-43.  T r i a l 2:  Sample means and standard deviations  (3 bags/sample and 7 judges) of scores f o r flavour of cooked prawn meat of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  6.67±0.94  6.67±0.94  6.6710.94  4  5.1712.61  5.1711.95  5.1711.67  8  3.8312.03  5.6011.20  5.0011.79  12  2.5011.38  4.6712.21  4.5011.50  214  Table B-44.  T r i a l 2:  Sample means and standard deviations  (3 bags/sample and 7 judges) of scores for texture o f cooked prawn meat of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  6.67±1.25  6.67±1.25  6.67±1.25  4  6.33±1.80  5.17±2.11  6.00±1.41  8  6.20±1.72  4.80±1.47  5.33±1.97  12  5.3311.80  6.33±1.60  5.0012.08  215  Table B-45.  T r i a l 2:  Sample means and standard deviations  (3 bags/sample and 7 judges) of overall sensory scores of the c o n t r o l , the CMAP, and the NMAP prawns on indicated sampling days  DAY  CONTROL  CMAP  NMAP  0  6.5010.92  6.5010.92  6.5010.92  4  5.6711.83  5.4711.30  5.7511.07  8  4.4511.06  5.0411.10  5.3110.43  12  3.4211.28  4.8111.52  4.4310.97  

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